Induced Plant Resistance to Herbivory

INDUCED PLANT RESISTANCE TO HERBIVORY Induced Plant Resistance to Herbivory Edited by Andreas Schaller University of...

Author: Andreas Schaller

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INDUCED PLANT RESISTANCE TO HERBIVORY

Induced Plant Resistance to Herbivory Edited by

Andreas Schaller University of Hohenheim, Stuttgart, Germany

Editor Andreas Schaller University of Hohenheim Stuttgart, Germany

ISBN: 978-1-4020-8181-1

e-ISBN: 978-1-4020-8182-8

Library of Congress Control Number: 2007941936 c 2008 Springer Science+Business Media B.V. No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.

Cover pictures showing Pieris brassicae caterpillars, the parasitic wasp Cotesia glomerata, and a parasitized Manduca sexta larva were taken by Hans Smid and Tibor Bukovinszky (http: www.bugsinthepicture.com/), and Johannes Stratmann (University of South Carolina). Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

In Memoriam Clarence A. (Bud) Ryan

Bud Ryan left us on October 7th, 2007. His sudden passing away is felt deeply by his family and friends. Bud has left us with a flourishing field of research but we must now continue along this road without him. Throughout his long career Bud gave the community many startling insights into nature. One of the first milestones in the long and unerring path to reveal the invisible secrets of the plant defense mechanism was the discovery, published in 1972, of wound-inducible proteinase inhibitors in potato. Much of Bud’s career was spent finding out how these proteins functioned in defense, how they were made, and how their genes were regulated. Constantly incorporating new ideas and technologies, Bud and his collaborators brought to light the first peptide hormone in plants (systemin) and found that jasmonates regulate proteinase inhibitor gene expression. These and other achievements initiated much if not most of the ongoing work in trying to understand the wound response in plants. All those who joined him in this endeavour were touched by his vision, his modesty, his strong sense of humour, and, above all, by his friendship. I personally feel the void he has left and know that this feeling is shared by my colleagues. Lausanne, Switzerland, October 18th, 2007 Edward E. Farmer

Photo: Bud Ryan (right) with his closest colleague Gregory Pearce in 1992. The image shows Bud on one of his frequent visits to the greenhouse to examine tomato plants engineered for altered defense responses. v

Contents

In Memoriam Clarence A. (Bud) Ryan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Section I Basic Concepts of Plant Defense Against Insect Herbivores 1 Direct Defenses in Plants and Their Induction by Wounding and Insect Herbivores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gregg A. Howe and Andreas Schaller

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2 Herbivore-Induced Indirect Defense: From Induction Mechanisms to Community Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Maaike Bruinsma and Marcel Dicke 3 Induced Defenses and the Cost-Benefit Paradigm . . . . . . . . . . . . . . . . . . 61 Anke Steppuhn and Ian T. Baldwin Section II Induced Direct Defenses

Part A Anatomical Defenses 4 Leaf Trichome Formation and Plant Resistance to Herbivory . . . . . . . 89 ˚ Peter Dalin, Jon Agren, Christer Björkman, Piritta Huttunen and Katri Kärkkäinen 5 Resistance at the Plant Cuticle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Caroline Müller vii

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Contents

6 Wound-Periderm Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Idit Ginzberg 7 Traumatic Resin Ducts and Polyphenolic Parenchyma Cells in Conifers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Paal Krokene, Nina Elisabeth Nagy and Trygve Krekling Part B Production of Secondary Metabolites 8 Insect-Induced Terpenoid Defenses in Spruce . . . . . . . . . . . . . . . . . . . . . 173 Jörg Bohlmann 9 Phenylpropanoid Metabolism Induced by Wounding and Insect Herbivory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Mark A. Bernards and Lars B˚astrup-Spohr 10 Defense by Pyrrolizidine Alkaloids: Developed by Plants and Recruited by Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Thomas Hartmann and Dietrich Ober Part C Anti-nutritional Enzymes and Proteins 11 Plant Protease Inhibitors: Functional Evolution for Defense . . . . . . . . 235 Maarten A. Jongsma and Jules Beekwilder 12 Defensive Roles of Polyphenol Oxidase in Plants . . . . . . . . . . . . . . . . . . 253 C. Peter Constabel and Raymond Barbehenn 13 Action of Plant Defensive Enzymes in the Insect Midgut . . . . . . . . . . . 271 Hui Chen, Eliana Gonzales-Vigil and Gregg A. Howe 14 Plant Lectins as Part of the Plant Defense System Against Insects . . . 285 Els J.M. Van Damme Section III Defense Signaling

Part A Activation of Plant Defenses 15 Systemins and AtPeps: Defense-Related Peptide Signals . . . . . . . . . . . . 313 Javier Narváez-Vásquez and Martha L. Orozco-Cárdenas

Contents

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16 MAP Kinases in Plant Responses to Herbivory . . . . . . . . . . . . . . . . . . . . 329 Johannes Stratmann 17 Jasmonate Biosynthesis and Signaling for Induced Plant Defense against Herbivory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 Andreas Schaller and Annick Stintzi Part B Signals Between Plants and Insects 18 Caterpillar Secretions and Induced Plant Responses . . . . . . . . . . . . . . . 369 Gary W. Felton 19 Fatty Acid-Derived Signals that Induce or Regulate Plant Defenses Against Herbivory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 James H. Tumlinson and Juergen Engelberth 20 Aromatic Volatiles and Their Involvement in Plant Defense . . . . . . . . . 409 Anthony V. Qualley and Natalia Dudareva 21 Ecological Roles of Vegetative Terpene Volatiles . . . . . . . . . . . . . . . . . . . 433 Jörg Degenhardt Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 Taxonomic Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457

Contributors

˚ Jon Agren Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, SE-752 36 Uppsala, Sweden [email protected] Ian T. Baldwin Max Planck Institute for Chemical Ecology, Department of Molecular Ecology, D-07745 Jena, Germany [email protected] Raymond Barbehenn Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48105, USA [email protected] Lars B˚astrup-Spohr Freshwater Biological Laboratory, University of Copenhagen, DK-3400 Hillerød, Denmark [email protected] Jules Beekwilder Plant Research International B.V., Wageningen University and Research Center, 6700 AA Wageningen, The Netherlands [email protected] Mark A. Bernards Department of Biology, The University of Western Ontario, London, ON, Canada N6A 5B7 [email protected] Christer Björkman Department of Ecology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden [email protected] xi

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Contributors

Jörg Bohlmann Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 [email protected] Maaike Bruinsma Laboratory of Entomology, Wageningen University, 6709 PD Wageningen, The Netherlands [email protected] Hui Chen DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA [email protected] C. Peter Constabel Centre for Forest Biology and Department of Biology, University of Victoria, Victoria, BC, Canada V8W 3N5 [email protected] Peter Dalin Marine Science Institute, University of California at Santa Barbara, CA 93106-6150, USA [email protected] Jörg Degenhardt Department of Biochemistry, Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany [email protected] Marcel Dicke Laboratory of Entomology, Wageningen University, 6709 PD Wageningen, The Netherlands [email protected] Natalia Dudareva Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA [email protected] Juergen Engelberth Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA [email protected] Gary W. Felton Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA [email protected]

Contributors

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Idit Ginzberg Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel [email protected] Eliana Gonzales-Vigil DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA [email protected] Thomas Hartmann Institute of Pharmaceutical Biology, Technical University of Braunschweig, D-38106 Braunschweig, Germany [email protected] Gregg A. Howe DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA [email protected] Piritta Huttunen Department of Biological and Environmental Sciences, University of Jyväskylä, 40350 Jyväskylä, Finland [email protected] Maarten A. Jongsma Plant Research International B.V., Wageningen University and Research Center, 6700 AA Wageningen, The Netherlands [email protected] Katri Kärkkäinen The Finnish Forest Research Institute, Muhos Research Unit, 91500 Muhos, Finland [email protected] Trygve Krekling Department of Plant and Environmental Sciences, Norwegian University of Life ˚ Norway Sciences, N-1432 As, [email protected] Paal Krokene ˚ Norway Norwegian Forest and Landscape Institute, N-1432 As, [email protected] Caroline Müller Department of Chemical Ecology, University of Bielefeld, D-33615 Bielefeld, Germany [email protected]

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Contributors

Nina Elisabeth Nagy ˚ Norway Norwegian Forest and Landscape Institute, N-1432 As, [email protected] Javier Narváez-Vásquez Department of Botany and Plant Sciences, University of California Riverside, Riverside, CA 92521, USA [email protected] Dietrich Ober Institute of Botany, University of Kiel, D-24098 Kiel, Germany [email protected] Martha L. Orozco-Cárdenas Plant Transformation Research Center, University of California Riverside, Riverside, CA 92521, USA [email protected] Anthony V. Qualley Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA [email protected] Andreas Schaller University of Hohenheim, Institute of Plant Physiology and Biotechnology, D-70599 Stuttgart, Germany [email protected] Anke Steppuhn Max Planck Institute for Chemical Ecology, Department of Molecular Ecology, D-07745 Jena, Germany [email protected] Annick Stintzi Institute of Plant Physiology and Biotechnology, University of Hohenheim, D-70599 Stuttgart, Germany [email protected] Johannes Stratmann Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA [email protected] James H. Tumlinson Center for Chemical Ecology, Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA [email protected]

Contributors

Els J.M. Van Damme Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, 9000 Gent, Belgium [email protected]

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Introduction

The class Insecta with more than one million described species is the most diverse group of animals on Earth and outnumbers all other forms of life. Almost half of all existing insect species are herbivores, i.e. they feed on living plants. The abilitity of plants to persist in such a hostile environment relies on evolved resistance systems allowing them to escape herbivores in time or in space, to confront herbivores directly by affecting host plant preference or reproductive success, or to fight herbivores indirectly by association with other species. Plant defenses against insect herbivores were long known to change in evolutionary time and may even change during the lifetime of an individual plant. Until recently however, plant defenses were generally assumed to be constitutively expressed, i.e. independent from herbivore attack. This view changed in 1972 when Green and Ryan reported that wounding by Colorado potato beetles causes the rapid accumulation of proteinase inhibitors in potato and tomato leaves. As inducible resistance factors which interfere with the digestive system of leaf-consuming insects, the proteinase inhibitors were suggested to defend the plants against herbivores. In the 35 years following the initial discovery by Green and Ryan, many more traits have been identified that are induced by wounding or herbivory. The full breadth of induced responses became apparent with the advent of novel techniques for transcriptome analysis, revealing large-scale changes in gene expression in response to herbivory. In fact, many of this book’s chapters focus on herbivoreinduced responses including the results of transcriptome analyses, and how they relate to plant defense. At this point, a cautionary note is required: Inducibility of a certain gene (or any given trait) is per se not sufficient evidence for a function in resistance or plant defense. Adopting the definitions advocated by R. Karban J.H. Myers, A.A. Agrawal, and I.T. Baldwin, the term ‘induced resistance’ refers to induced changes in preference, performance, or reproductive success of the attacker. Therefore, to test for a role in resistance, it is necessary to compare herbivore preference and/or performance on plants that differ in the trait of interest, which can easily be done in a laboratory setting. Induced resistance is thus defined from the point of view of the herbivore, and it does not necessarily benefit the plant. For example, the investment in induced resistance may be larger than the benefit form reduced herbivore damage, or induced herbivore resistance may render the plant

A. Schaller (ed.), Induced Plant Resistance to Herbivory, C Springer Science+Business Media B.V. 2008

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Introduction

more susceptible to other stresses. Defense, on the other hand is defined from the plant’s perspective, and it implies a benefit for the plant. Only when they result in a gain in plant fitness are induced traits considered to contribute to plant defense. Therefore, to demonstrate a role in plant defense, field studies are required that reveal differences in fitness for plants that differ in the trait of interest. Only very few herbivore-induced responses have actually been shown to result in a fitness benefit for the plant. Nevertheless, the term ‘defense’ is frequently used by researchers in the field and also by the authors of this book to describe induced changes in plant architecture, metabolism, or physiology that are assumed to minimize the negative impact of herbivory. Two types of defense are commonly distinguished (Table 1). Defense is called direct if induced responses affect the interaction of the herbivore and its host plant directly. It relies on resistance factors that by themselves or in combination impact on the preference of insect herbivores during host plant selection (antixenosis), or the performance of the insect on its host plant (antibiosis). Resistance factors include morphological features of the plant which act as physical barriers to ward off invading pests, and the chemical constitution of the plant which is the primary determinant for host plant selection. As an additional component of direct defense, plants may develop tolerance, allowing them to support a herbivore population similar to that on a susceptible host but without the concomitant reduction in plant fitness. Underlying tolerance are traits related to plant morphology and to the production or allocation of resources which – similar to resistance traits – may be constitutive or inducible by herbivory (Table 1). For indirect defense plants rely on natural enemies of their herbivores. Morphological and chemical features support the presence, abundance, and effectiveness of predators or parasites of herbivorous insects, and may thus serve as resistance factors. Examples include hollow thorns (domatia) that provide shelter, and extrafloral nectar that is produced as a food source for ants which, in a mutualistic relationship with myrmecophilic plants, protect their hosts from herbivores. Furthermore, in response to herbivory, plants may produce odors that attract predators or parasitoids of herbivores and guide them to their prey. Table 1 includes carnivorous enemies of insect herbivores as ‘ecological resistance factors’ that are recruited by the plant for indirect defense.

Table 1 Classification of defensive traits. Resistance factors can be categorized as physical (including morphological and structural features of the plant), biochemical (e.g., toxic or anti-nutritive metabolites and proteins), or ecological (i.e., involving other species). They differ with respect to their modes of expression (constitutive or inducible) and action (in direct or indirect defense) Type of trait Physical

Mode of expression

Mode of action

Constitutive

Direct defense (antixenosis, antibiosis, tolerance)

(Bio)chemical Induced Ecological

Indirect defense (tritrophic interactions)

Introduction

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Direct and indirect mechanisms of plant defense against herbivores, and the raison d’être for inducibility as opposed to constitutive expression of defense are introduced in Section I of this book. These introductory chapters provide the background for the subsequent more focussed discussion of individual resistance factors in Section II, and the signals and signaling mechanisms for the induction of direct and indirect defenses in Section III. With emphasis on plant responses that are induced by wounding or herbivory, the progress of research is summarized in a field that continues to be highly dynamic, even 35 years after the initial discovery of proteinase inhibitor accumulation as a wound-induced anti-nutritive defense by Ryan in 1972.

Section I

Basic Concepts of Plant Defense Against Insect Herbivores

Chapter 1

Direct Defenses in Plants and Their Induction by Wounding and Insect Herbivores Gregg A. Howe and Andreas Schaller

Resistance factors for direct plant defense against herbivorous insects comprise plant traits that negatively affect insect preference (host plant selection, oviposition, feeding behavior) or performance (growth rate, development, reproductive success) resulting in increased plant fitness in a hostile environment. Such traits include morphological features for physical defense, like thorns, spines, and trichomes, epicuticular wax films and wax crystals, tissue toughness, as well as secretory structures and conduits for latices or resins. They also include compounds for chemical defense, like secondary metabolites, digestibility reducing proteins, and antinutritive enzymes. All these traits may be expressed constitutively as preformed resistance factors, or they may be inducible and deployed only after attack by insect herbivores. The induction of defensive traits is not restricted to the site of attack but extends to non-infested healthy parts of the plants. The systemic nature of plant responses to herbivore attack necessitates a long-distance signaling system capable of generating, transporting, and interpreting alarm signals produced at the plant–herbivore interface. Much of the research on the signaling events triggered by herbivory has focused on tomato and other solanaceous plants. In this model system, the peptide systemin acts at or near the wound site to amplify the production of jasmonic acid. Jasmonic acid or its metabolites serve as phloem-mobile long-distance signals, and induce the expression of defense genes in distal parts of the plant. In this chapter, we will provide an overview of physical and chemical defense traits, and review the signaling mechanisms that account for their inducible expression after insect attack.

1.1 Introduction Plants, flowering plants in particular, exhibit a tremendous diversity in size and shape, ranging from just a few millimeters in the tiny duckweeds to almost 100 A. Schaller University of Hohenheim, Institute of Plant Physiology and Biotechnology, D-70599 Stuttgart, Germany e-mail: [email protected]


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G.A. Howe, A. Schaller

meters in giant eucalyptus trees. Some may complete their life cycle in a few weeks, while others live thousands of years. The amazing diversity results from the adaptation to different, oftentimes hostile environments, as exemplified by the early evolution of land plants. The colonization of land by plants, dating back some 480 million years according to fossil records (Kenrick and Crane 1997), marks the beginning of an evolutionary success story, with flowering plants now occupying every habitat on Earth except the regions surrounding the poles, the highest mountaintops, and the deepest oceans (Soltis and Soltis 2004). The colonization of land was a major event in the history of plant life, and at the same time, paved the way for the explosive evolution of terrestrial ecosystems. Despite the vulnerability of plants as sessile organisms to adverse biotic and abiotic conditions, they actually dominate over much of the land surface. This apparent success of flowering plants relies on the evolved ability to persist in unfavorable and variable environments by virtue of effective resistance systems that are based on a combination of physical, chemical, and developmental features (Schoonhoven et al. 2005). It was recognized by Stahl in 1888 that the great diversity of mechanical and chemical ‘means of protection of plants were acquired in their struggle for existence within the animal world’ leading to the conclusion that ‘the animal world [. . .] deeply influenced not only their morphology but also their chemistry’ (Stahl 1888; Fraenkel 1959). Hence, not only thorns and spines as morphological resistance traits, but also the bewildering variety of plant secondary chemicals attest to the selective pressure exerted by phytophagous animals (Fraenkel 1959; Ehrlich and Raven 1964). It was later discovered that induced expression of resistance traits increases plant fitness in environments that harbor a variety of plant parasites. The inducibility of plant resistance was first reported for fungal and bacterial pathogens in the early 1900s (Karban and Kuc 1999) and, much later, inducible defenses were shown to exist also against insect herbivores. In their seminal paper of 1972, Green and Ryan demonstrated that tomato and potato plants accumulate inhibitors of trypsin and chymotrypsin-like serine proteinases throughout their aerial tissues, as a direct consequence of insect-mediated damage or mechanical wounding (Green and Ryan 1972). Proteinase inhibitors are present constitutively in high concentrations in plant storage organs, and a possible function as protective agents against insects was discussed at that time (Lipke et al. 1954; Applebaum and Konijn 1966). Green and Ryan suggested that the expression of proteinase inhibitors may be regulated in leaves to make the plant less palatable and perhaps lethal to invading insects. The accumulation of proteinase inhibitors in aerial tissues was proposed to constitute an inducible defense system, directly affecting the performance of leaf-consuming insects by starving them of nutrients, thus resulting in enhanced plant resistance against herbivory (Green and Ryan 1972). It is now clear that the nutritional quality of the foliage is an important determinant of herbivore growth and development (Painter 1936; Berenbaum 1995; Schoonhoven et al. 2005) and anti-nutritional defense as part of the plant’s arsenal for induced resistance is well accepted (Rhoades and Cates 1976; Felton 2005). Thirty-five years of research following the initial discovery by Ryan and coworkers established plant resistance against insect herbivores as a highly dynamic

1 Herbivore-Induced Direct Defense

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process. In addition to the proteinase inhibitors, many more inducible factors have been identified which contribute to direct defense and which have the potential to enhance host plant fitness after herbivore attack. These are aspects that will be introduced in this chapter to provide the background for a more detailed discussion of the defensive role of individual proteins in the subsequent, more focused chapters of this volume. Another aspect of induced resistance that has fascinated researchers since the seminal Green-and-Ryan-paper is the systemic nature of the response: defense proteins accumulate not only at the site of wounding but also systemically in unwounded tissues of the infested plant. Obviously, a signal must be generated locally as a consequence of insect feeding which is then propagated throughout the plant, and able to induce the expression of defense proteins at distant sites (Green and Ryan 1972; Ryan and Moura 2002). Our current understanding of systemic wound signaling for direct defense will also be summarized here.

1.2 Inducible Resistance Factors for Direct Defense Since the initial observation of proteinase inhibitor accumulation in wounded tomato and potato plants, inducibility by herbivory has been shown for a large number of other potential resistance factors (Walling 2000; Gatehouse 2002). In the light of recent studies analyzing induced responses at the level of the entire transcriptome, we now begin to appreciate the full breadth and highly dynamic nature of plantinsect interactions. Numerous studies have shown that herbivory causes large-scale changes in gene expression (Cheong et al. 2002; Delessert et al. 2004; Reymond et al. 2004; Smith et al. 2004; Voelckel and Baldwin 2004; Zhu-Salzman et al. 2004; De Vos et al. 2005; Schmidt et al. 2005; Ralph et al. 2006b; Thompson and Goggin 2006; Broekgaarden et al. 2007). In hybrid poplar, for example, it is estimated that 11% of the transcriptome is differentially regulated by insect feeding (Ralph et al. 2006a). However, inducibility of a certain gene or enzyme per se is not sufficient evidence for a function in plant defense. Whereas the potential contribution of a given trait to plant resistance can be readily tested in a laboratory setting by comparing herbivore preference and/or performance on plants that differ in the trait of interest, a role in plant defense implies that expression of the resistance trait is associated with a gain in plant fitness; such associations must ultimately be demonstrated in field experiments that simulate ‘real world’ conditions (Karban and Myers 1989). A further requisite for the evolution of inducible defense systems is heritable variation in the degree of inducibility (Karban and Myers 1989; Agrawal 1999). Genetic variation has frequently been observed in natural populations, e.g., for physical (trichome density) or chemical (glucosinolate content) resistance characters in Arabidopsis, and both traits are associated with fitness costs (Mauricio 1998). For induced resistance traits, however, a fitness benefit has been demonstrated in only a few cases. One example is radish plants that were induced to accumulate higher levels of glucosinolates and to produce trichomes at increased density. Compared to control plants, these induced plants exhibited both increased resistance to herbivory

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and increased seed mass (a correlate of lifetime fitness). This experiment confirmed a role in direct defense for trichomes and glucosinolates as inducible physical and chemical resistance factors, respectively (Agrawal 1998, 1999). Likewise, in Nicotiana attenuata, the induced production of nicotine as a chemical resistance factor was associated with metabolic costs, but provided a fitness benefit when plants were under attack by herbivores (Baldwin 1998; see also Steppuhn and Baldwin this volume). Although these findings should not be generalized and a defensive role should not be assumed for all plant responses to wounding and herbivory, the prevalence of inducible resistance traits in present day plant-herbivore systems implies that such responses are likely the result of natural selection imposed by insect herbivores during evolution. Any plant trait that interferes with host plant selection, oviposition, or feeding of an insect herbivore is a potential resistance factor and may further contribute to plant defense. Most prominent among these traits are morphological features and the chemical composition of the plant, both of which have long been recognized as constitutive resistance characters (Stahl 1888; Fraenkel 1959), and were also the focus of initial studies on inducible resistance to insect herbivores. In this chapter, we provide a brief overview of inducible factors that lead to enhanced resistance through direct effects on insect preference or performance. The traditional distinction between plant defense traits that are either morphological or chemical is used throughout this volume (see also Table 1 on page 2 in the Introduction). It is important to realize, however, that this classification is often arbitrary because any morphological feature is the manifestation of a genetically regulated biochemical process and, therefore, also chemical at its very basis.

1.2.1 Morphological Features for Physical Defense Insect herbivores from all feeding guilds must make contact with the plant surface in order to establish themselves on the host plant. It is therefore not surprising that physical and chemical features of the plant surface are important determinants of resistance. Epicuticular wax films and crystals cover the cuticle of most vascular plants. In addition to their important role in desiccation tolerance, they also increase slipperiness, which impedes the ability of many non-specialized insects to populate leaf surfaces. The physical properties of the wax layer as well as its chemical composition are important factors of preformed resistance (see Müller this volume). Whereas induced changes in wax production and surface chemistry have been observed, evidence for a role of the cuticle and epicuticular waxes in induced resistance is still scant. Wax biosynthesis and composition are known to vary during plant development, and the physico-chemical properties of the cuticle respond to changes in season and temperature (Müller this volume). Considering the ingenuity of plants in dealing with their offenders, it would thus be surprising if regulated production of wax on the leaf surface were not adopted to influence the outcome of plant-insect interactions. Other components of the plant surface that serve a role in constitutive defense include thorns and spines directed against mammalian herbivores, and hairs


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(trichomes) which are effective against insects (Myers and Bazely 1991; Schoonhoven et al. 2005). Non-glandular trichomes may serve as structural resistance factors preventing small insects from contacting the leaf surface or limiting their movement. Morphological and chemical resistance factors are combined in glandular trichomes. Glands produce substances which may repel insect herbivores or deter them from feeding (antixenosis), or immobilize them on the leaf surface. Quite interestingly, trichome density in some plant species increases in response to insect feeding, and therefore constitutes an inducible resistance trait. The defensive role of trichomes is discussed in more detail by Dalin et al. (this volume). Leaf toughness is an important physical factor for plant resistance, as it affects the penetration of plant tissues by mouthparts of piercing-sucking insects, and also increases mandibular wear in biting-chewing herbivores (Schoonhoven et al. 2005). Leaf toughness is frequently correlated with insect resistance and is a good predictor of herbivory rates (Bergvinson et al. 1995; Coley and Barone 1996; Howlett et al. 2001). Although leaf toughness is typically regarded as a physical character, this trait exemplifies the general difficulty in drawing clear distinctions between physical and chemical resistance factors. Cell wall reinforcement for enhanced leaf toughness results from the deposition of ‘chemicals’, including macromolecules such as lignin, cellulose, suberin, and callose, small organic molecules (e.g., phenolics), and even inorganic silica particles (Schoonhoven et al. 2005). Enhanced synthesis and/or deposition of these chemicals after wounding leads to induced physical resistance (McNaughton and Tarrants 1983; Bernards this volume; Ginzberg this volume). Another anatomical defense found in plants of diverse phyolgenetic origin is a network of canals such as lacticifers (latex-containing living cells) or resin ducts (resin-filled intercellular spaces) that store latex or resins under internal pressure. When the canal system is severed, the contents are exuded and may entrap or even poison the herbivore. Out of more than 50 plant families for which such defense systems have been described, the well-studied milkweeds (genus Asclepias in the family Asclepiadacea) may serve as an example. Milkweed latices coagulate upon exposure to air and immobilize small insect larvae. As an additional chemical resistance factor, the latex may contain large amounts of toxic cardenolides (Dussourd and Hoyle 2000; Agrawal 2004). Fascinatingly, many specialist herbivores that feed on milkweed or other latex-producing plants employ feeding strategies that block the flow of latex to intended feeding sites. Such feeding behavior has evolved independently in several phylogenetic lineages, and can be viewed as a counteradaptation of herbivores to circumvent latex-based plant defenses (Carroll and Hoffman 1980; Dussourd and Eisner 1987; Dussourd and Denno 1994). A widely appreciated and well-established form of anatomical protection are the resin-based defenses in conifers (Berryman 1972). The resin, which is a mixture of monoterpenes, sesquiterpenes, and diterpene resin acids, accumulates in resin ducts and related secretory structures. Stem-boring bark beetles and other insects that breach the resin duct system are expelled (‘pitched out’) from the bore hole by resin flow. Upon exposure to air, the highly volatile monoterpene fraction evaporates, leaving the insects trapped in the solidifying resin acids and the wound site

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sealed (Phillips and Croteau 1999; Trapp and Croteau 2001). Although this complex resin-based defense system in conifers is preformed, it is further induced in response to wounding. Among the inducible components of the system are terpene biosynthesis (Bohlmann this volume) and the formation of new resin ducts (Krokene et al. this volume). Finally, the wound healing process itself can be considered as a wound-induced anatomical trait for enhanced resistance. Efficient sealing of the wound is important to prevent water loss and opportunistic infections by bacterial and fungal pathogens at the site of tissue damage. Wound closure may involve extensive cell division and formation of wound callus (e.g., Guariguata and Gilbert 1996). In the case of plants with resin- and latex-based defenses, coagulation of the exudates may efficiently seal the wound site. More generally, a sealing cell layer is formed by infusion of antimicrobial and water-impermeable substances, including lignin and suberin (Rittinger et al. 1987). This may be followed by the induction of cell division and the formation of a periderm as a protective tissue that is impermeable to water and resistant to pathogens. Wound periderm formation and its potential contribution to plant defense are discussed in greater detail by Ginzberg (this volume).

1.2.2 Metabolites and Enzymes for Chemical Defense Plant chemicals that play a role in direct defense impair herbivore performance by one of two general mechanisms: these chemicals may reduce the nutritional value of plant food, or they may act as feeding deterrents or toxins. There has been considerable debate as to which of these two strategies is more important for host plant selection and insect resistance. An important part of this debate concerns the extent to which variation in the levels of primary and secondary metabolites has evolved as a plant defense (Berenbaum, 1995). Plant primary metabolism, which is shared with insects and other living organisms, provides carbohydrates, amino acids, and lipids as essential nutrients for the insect. Food quality is largely determined by the availability of these nutrients, and its importance for longevity, size, fecundity, and death rates in herbivorous insects has been recognized early on by Painter (1936). In addition, more than 100,000 plant compounds (i.e., secondary metabolites) have been identified with no apparent role in primary metabolism, and many of these have been regarded as expendable metabolic waste products. While many secondary metabolites are in fact expendable for primary metabolism, it is now widely accepted that they serve important ecological functions in the interaction of plants with their biotic and abiotic environment. According to the paradigm put forward by Fraenkel in his seminal paper in 1959, secondary metabolites in a given plant species may act both as repellents for generalist (polyphagous) insects and as attractants for specialist (monophagous) insects, and may thus be largely responsible for host range restriction (Fraenkel 1959). In addition to these allelochemical functions, secondary metabolites also act in multiple ways as toxins, feeding deterrents, as digestibility reducers or antinutritives,


13

as precursors for physical defense, and as volatiles in indirect defense (Bennett and Wallsgrove 1994; Karban and Baldwin 1997). Despite their diversity in structure, activity, and distribution in the plant kingdom, all secondary compounds are derived from universally available intermediates of primary metabolism, including sugar phosphates (erythrose 4-phosphate), acetyl-coenzyme A, and amino acids, and are conveniently classified according to their biosynthetic pathways as phenolics, terpenoids, and alkaloids. Each of these classes of compounds and their role in induced resistance are the focus of subsequent chapters of this volume. While the importance of secondary metabolites in plant defense remained undisputed for decades following Fraenkel’s landmark paper (Fraenkel 1959), the realization that some secondary metabolites (e.g., tannins and phenolics) exert anti-nutritive activity brought greater attention to the idea that food quality, nutritional value, and variation in primary metabolism may have evolved as a plant defense (Feeny 1970; Rhoades and Cates 1976; Berenbaum 1995). The relevance of nutritional quality as a resistance trait was further supported by Ryan and coworkers (Green and Ryan 1972) who showed that induced expression of serine proteinase inhibitors contributes to plant defense by interfering with the insect’s digestive processes, thus limiting the availability of essential amino acids. Following the landmark study of Green and Ryan, many workers reported that the overall chemical composition of the plant is greatly influenced by developmental and environmental parameters, including herbivory. Induced changes in plant chemistry involve the biosynthesis of a wide variety of secondary metabolites, including phenolics, terpenoids, alkaloids, cyanogenic glucosides, and glucosinolates (Karban and Baldwin 1997; Constabel 1999; Chapters 8–10, this volume). It was further shown that the induction of anti-nutritional proteins is not limited to serine proteinase inhibitors, but includes inhibitors of other classes of proteases, oxidative enzymes, amino acid-metabolizing enzymes, and lectins (Constabel 1999; Felton 2005). Such examples of protein-based defenses are further discussed in Chapters 11–14 of this volume.

1.2.3 Metabolic Reconfiguration to Shift from a Growthto a Defense-Oriented State The numerous anatomical and chemical changes associated with induced resistance require massive reprogramming of gene expression. For the quantitative analysis of large-scale changes in gene expression, novel techniques have been developed in recent years. Most notable among these approaches are microarray technologies for the identification of differentially expressed transcripts, and even more recently, techniques for high-throughput proteomic analysis (Kessler and Baldwin 2002; Kuhn and Schaller 2004; Giri et al. 2006; Lippert et al. 2007). With the advent of these techniques, it is now possible to obtain a relatively unbiased account of the plant’s response to herbivory. Many of the genes required for the expression of known resistance traits were in fact shown to be upregulated during

14


plant-insect interaction. Consistent with the activation of structural defenses, genes of general phenylpropanoid metabolism and monolignol biosynthesis, lignin polymerization, and cell wall fortification are induced by wounding or herbivory in hybrid poplar (Smith et al. 2004; Lawrence et al. 2006; Major and Constabel 2006; Ralph et al. 2006a), Sitka spruce (Ralph et al. 2006b), and Arabidopsis (Cheong et al. 2002; Delessert et al. 2004; Reymond et al. 2004). Likewise, the activation of chemical defenses is accompanied by the induction of genes involved in secondary metabolism, including phenolics, polyamine, and alkaloid biosynthesis in N. attenuata (Voelckel and Baldwin 2004; Schmidt et al. 2005; Giri et al. 2006), the genes for the formation of phenolics and terpenes in spruce and poplar (Ralph et al. 2006a, b), and phenolic metabolism and glucosinolate biosynthesis in Arabidopsis (Cheong et al. 2002; Reymond et al. 2004). These studies also confirmed the activation of genes for antidigestive and antinutritional defenses (e.g., proteinase inhibitors, oxidative enzymes, lectins), and for the signaling of the resistance response (e.g., jasmonic acid and ethylene biosynthesis, transcription factors). Notably, however, these genes represent only a fraction of the total wound-induced changes in gene activity. The insect-responsive transcriptome was estimated to comprise 10% of all transcripts, suggesting that massive reprogramming of gene expression is required to bring about a shift from growth-oriented to defense-oriented plant metabolism (Hui et al. 2003; Ralph et al. 2006a). The latter state involves the activation of genes for general stress responses (oxidative stress, dehydration stress, heat-shock proteins), protein turnover (e.g., proteases), and transport processes (e.g., aquaporins, lipid transfer proteins, ABC transporters, sugar and peptide transporters), as well as modulation of primary metabolism (carbohydrate and lipid metabolism, nitrogen assimilation), and downregulation of photosynthesis and chloroplast function. These changes in gene expression may reflect the herbivore-induced reallocation of resources from primary processes to defense (Voelckel and Baldwin 2004; Ralph et al. 2006b). Efficient mobilization of plant resources is likely to facilitate the expression of costly resistance traits, including the accumulation of defense proteins, the synthesis of secondary metabolites, and the formation of structural defenses. On the other hand, mobilization of resources may also contribute to plant tolerance of herbivory. Unlike resistance, tolerance does not affect herbivore preference or performance, but rather allows the host plant to minimize the fitness consequences of tissue loss. Tolerance and resistance are therefore viewed as alternative and complementary strategies for plant defense against insect herbivores (Karban and Myers 1989; Mauricio 2000; Weis and Franks 2006). Whereas tolerance is still not well-understood at the molecular level, it may include the mobilization of leaf carbon and nitrogen that is threatened by herbivory, and temporary storage of these resources for later regrowth. The induction of protein turnover, lipid and carbohydrate metabolism, and transport functions observed in microarray studies (see above) may thus be equally relevant for both tolerance and induced expression of resistance traits. Temporary storage of resources occurs in organs that are less susceptible to herbivory, e.g., the root system. Indeed, a change in sink-source relations was observed in N. attenuata after simulated herbivore attack, resulting in increased allocation of sugars to roots


15

and enhanced tolerance (Schwachtje et al. 2006). Likewise, the induction of vegetative storage proteins frequently observed in response to wounding (Staswick 1994; Christopher et al. 2004; Reymond et al. 2004; Major and Constabel 2006) may allow the plant to buffer mobilized resources for later use in re-growth. Remarkably, such a role as interim storage or temporary protein depot had already been suggested for proteinase inhibitor I in tomato and potato plants, the first protein shown to be systemically induced by herbivory (Ryan and Huisman 1969; Green and Ryan 1972).

1.3 Systemic Signaling for Induced Direct Defense An important feature of many wound-induced direct defense responses is their occurrence in undamaged tissues located far from the site of wounding. Woundinducible serine proteinase inhibitors (PIs) represent one of the best examples of a systemically induced defense response. In tomato plants, PI genes are expressed in distal leaves within 1–2 hrs after insect attack or mechanical wounding (Ryan 2000; Strassner et al. 2002). The rapid and systemic nature of this response is analogous to vertebrate immune responses in which endocrine signals are delivered to target tissues via the circulatory system (Bergey et al. 1996). However, because plants lack mobile defender cells, systemic signals must be transmitted long distances via mechanisms that are specific to plants (Malone 1996; León et al. 2001; Schilmiller and Howe 2005). Ryan’s pioneering work on systemic wound signaling inspired generations of plant biologists to investigate the underlying mechanisms of this fascinating response. The widespread occurrence of systemic defense responses in the plant kingdom implies the existence of common mechanisms to generate, transport, and perceive alarm signals that are generated at the site of tissue damage. Wound-inducible PIs in tomato and other solanaceous plants have been widely used as a model system in which to study the molecular mechanism of systemic wound signaling. Green and Ryan (1972) proposed that chemical signals produced at the wound site travel through the plant and activate PI expression in undamaged leaves. Identification of these signaling compounds was facilitated by a simple bioassay in which test solutions (e.g., containing an elicitor) are supplied to tomato seedlings through the cut stem, followed by measurement of PI accumulation in the leaves. Extensive use of this assay led to the discovery of several distinct classes of PI-inducing compounds, including cell-wall-derived oligogalacturonides (OGAs), systemin, jasmonic acid (JA), and hydrogen peroxide (Ryan 2000; Gatehouse 2002). Physical signals (e.g., hydraulic forces and electrical signals) generated by tissue damage have also been implicated in the systemic signaling process (Wildon et al. 1992; Malone 1996). Currently, a major challenge is to determine how these diverse signals interact with one another to promote intercellular communication across long distances. Farmer and Ryan (1992) established the current paradigm that extracellular signals such as OGAs and systemin (so-called primary wound signals), generated in response to wounding, trigger the intracellular production of JA via the

16


octadecanoid pathway and that JA, in turn, activates the expression of defensive genes. Wound-induced production of OGAs is catalyzed by a family of polygalacturonases (PGs) that are expressed in various plant tissues (Bergey et al. 1999). OGAs are relatively immobile in the plant vascular system and thus are thought to act as local mediators. However, because PG activity is induced systemically in response to wounding, OGAs could also amplify defense responses in undamaged leaves (Ryan 2000). OGA-mediated signal transduction may result from direct physical effects of these compounds on the plasma membrane or may involve specific receptors (Navazio et al. 2002). Systemin was the first bioactive peptide discovered in plants (Pearce et al. 1991). This 18-amino-acid peptide is derived from proteolytic cleavage of a larger precursor protein, prosystemin. When used in the tomato seedling bioassay, systemin is >10, 000-fold more active than OGAs in inducing PI expression. Several lines of evidence indicate that systemin serves a key role in induced defense responses in tomato. For example, transgenic plants expressing an antisense prosystemin (Prosys) cDNA are deficient in wound-induced systemic expression of PIs and, as a consequence, are more susceptible to insect herbivores (McGurl et al. 1992; Orozco-Cárdenas et al. 1993). Overexpression of prosystemin from a 35S::Prosys transgene constitutively activates PI expression in the absence of wounding, thereby conferring enhanced resistance to herbivores (McGurl et al. 1994; Li et al. 2002; Chen et al. 2005). Forward genetic analysis has shown that genes required for systemin-mediated signaling are essential for wound-induced expression of PI and other defense-related genes (Howe and Ryan 1999; Howe 2004). Thus, wounding and systemin activate defense genes through a common signaling pathway. Transcriptional activation of defense genes in response to systemin requires the biosynthesis and subsequent action of JA (Farmer and Ryan 1992; Howe, 2004). The systemin signaling pathway is initiated upon binding of the peptide to a 160-kDa plasma membrane-bound receptor (SR160) that was identified as a member of the leucine-rich repeat (LRR) receptor-like kinase family of proteins (Scheer and Ryan 1999, 2002). Binding of systemin to the cell surface is associated with several rapid signaling events, including increased cytosolic Ca2+ levels, membrane depolarization, and activation of a MAP kinase cascade (Felix and Boller 1995; Stratmann and Ryan 1997; Moyen et al. 1998; Schaller and Oecking 1999). The precise mechanism by which systemin activates JA synthesis remains to be determined. There is evidence indicating that a systemin-regulated phospholipase A2 activity in tomato leaves releases linolenic acid, a JA precursor, from lipids in the plasma membrane (Farmer and Ryan 1992; Narváez-Vásquez et al. 1999). Alternatively, the role of a chloroplast-localized phospholipase A1 in JA biosynthesis (Ishiguro et al. 2001) raises the possibility that systemin perception at the plasma membrane is coupled to the activation of a similar lipase in the chloroplast. JA synthesized in response to systemin, OGAs, and wounding acts in concert with ethylene (O’Donnell et al. 1996) and hydrogen peroxide (Orozco-Cárdenas et al. 2001; Sagi et al. 2004) to positively regulate the expression of downstream target genes.


17

1.3.1 Genetic Analysis of the Wound Response Pathway in Tomato Genetic analysis provides a powerful approach to identify components of the systemic wound response pathway. The robust nature of wound-induced PI expression in tomato, together with facile assays for PIs and other biochemical markers (e.g., polyphenol oxidase) of the response, has been exploited for this purpose. Forward genetic screens identified mutants that are defective in PI expression in response to mechanical wounding or treatment with methyl-JA (MeJA) (Howe 2004; Li et al. 2004). Additional screens have been conducted to identify mutations that suppress the inductive effects of the 35S::Prosys transgene (Howe and Ryan 1999). These screens have yielded numerous mutants that are deficient in wound-induced systemic expression of defensive genes. That most of these mutants display altered resistance to arthropod herbivores and various pathogens demonstrates the importance of induced responses to plant protection (Howe 2004). Map-based cloning and candidate gene approaches were used to identify genes defined by forward genetic analysis. The spr2 and acx1 mutants, which were generated by ethylmethane sulfonate mutagenesis, are defective in genes required for JA biosynthesis. Spr2 encodes a plastidic ␻-3 fatty acid desaturase that converts linoleic acid to the JA precursor linolenic acid (Li et al. 2003). ACX1 encodes a peroxisomal acyl-CoA oxidase that catalyzes the first step in the ␤-oxidation stage of JA synthesis (Li et al. 2005). The jasmonate insensitive1 ( jai1) mutant harbors a deletion in the tomato ortholog of the Arabidopsis Coronatine insensitive1 (Coi1) gene (Li et al. 2004). Coi1 encodes an F-box protein that is essential for expression of jasmonate-responsive genes, including many wound-responsive genes involved in anti-insect defense (Xie et al. 1998). Reverse genetic strategies identified several additional wound response mutants of tomato. For example, transgenic lines specifically engineered for defects in JA biosynthesis (Stenzel et al. 2003), ethylene synthesis (O’Donnell et al. 1996), ABA signaling (Carrera and Prat 1998), and ROS production (Sagi et al. 2004) are impaired in wound-induced systemic PI expression and other defense responses.

1.3.2 Jasmonate Performs a Key Role in Systemic Wound Signaling Despite significant progress in the identification of genes that regulate systemic defense responses, relatively little is known about the specific role of these components in the long-distance signaling pathway. In theory, genes required for the systemic response could play a role in production of the mobile signal, translocation of the signal from damaged to undamaged leaves, signal perception by target cells in distal leaves, or subsequent signaling steps leading to expression of target genes. Classical grafting techniques provide a powerful approach to determine whether a particular mutant is defective in the production of the systemic (i.e., graft-transmissible) wound signal or the recognition of that signal in responding leaves (Li et al. 2002). Reciprocal grafting experiments performed with the

18


JA-insensitive jai1 mutant showed that jasmonate perception (i.e., COI1) is essential for recognition of the mobile signal in distal responding leaves (Fig. 1.1Ad). These studies also suggest that the mobile signal is produced in the absence of COI1 (Fig. 1.1Ac). Experiments conducted with JA biosynthetic mutants (e.g., acx1) showed that production of the graft-transmissible signal depends on JA biosynthesis in wounded tissues (Fig. 1.1Bc). The ability of JA-deficient scions to express PIs in response to a signal emanating from wild-type rootstock leaves further indicated that de novo JA synthesis is likely not necessary for recognition of the mobile signal in the responding leaves (Fig. 1.1Bd). Based on these collective studies, it was proposed that JA (or a JA derivative) is a critical component of the systemic signal (Schilmiller and Howe 2005). These findings are also consistent with DNA microarray studies showing that local and systemic tissues undergo distinct signaling events (Strassner et al. 2002). The plant vascular system is involved in long-distance trafficking of a wide range of signaling compounds (Lucas and Lee 2004). Recent studies provide direct evidence that jasmonates are transported in the phloem (Fig. 1.2). For example, several JA biosynthetic enzymes are located in the companion cell-sieve element complex of the vascular bundle (Hause et al. 2003; Wasternack 2007). This observation is supported by the occurrence of JA in phloem bundles from Plantago major (Hause et al. 2003) and the preferential accumulation of jasmonates in the tomato leaf midrib (Stenzel et al. 2003). The hypothesis that the systemic signal is translocated in the phloem is further supported by the fact that wound-induced systemic responses are strongly enhanced by the strength of vascular connections between wounded and responding leaves (Davis et al. 1991; Schittko and Baldwin 2003). The rate of movement of the endogenous signal in tomato plants is estimated between 1 and 5 cm/hr (Schilmiller and Howe 2005). The ability of the phloem to transport small molecules at rates up to 40 cm/hr (Fisher 1990) could readily accommodate such a signal. Because systemic PI expression is mediated by a signal traveling within the plant rather than a signal diffusing through the atmosphere (Farmer and Ryan 1992), it is unlikely that volatile MeJA released at the wound site is a causal factor for systemic PI expression in tomato. The idea that JA (or a JA derivative) functions as a mobile wound signal implies that JA synthesized in damaged leaves is transported to distal undamaged leaves. In tomato and other dicots, however, systemic increases in JA levels in response to mechanical damage are generally very low (i.e., 50% 50%

Leaves Shoots

Leaves

Leaves

Leaves

Shoots Leaves Leaves Leaves Leaves

Tissue damaged

5%–25% >50%

3%

10%

5%

no info. 20%–25% 50% 100% 5%

Amount

The life history of the different plant species is indicated as (p) for perennial, or (a) for annual

Artificial

Salix cinerea (p)

1

Livestock Lepidoptera Lepidoptera Coleoptera Coleoptera

Defoliation treatment Damage Environment

Cnidoscolus texanus (p) Lepidium virginicum (a) Raphanus raphanistrum (a) Salix borealis (p) Salix cinerea (p)

Table 4.1 (continued)

50%

no induction

no induction

26%

23%–106%

13% 140%–790%

83%–186%

93%

no induction

38% 60% 25%–100% 51%–300% 72%

5–7 weeks

5–7 weeks

5–7 weeks

5–7 weeks

10 weeks

3 weeks 10 weeks

3 weeks

3 weeks

3 weeks

no info. 2 weeks 3 weeks next year 3 weeks

Increase in trichome density Magnitude Timing

Pollard (1986) Agrawal (2000) Agrawal (1999) Zvereva et al. (1998) Dalin and Björkman (2003) Dalin and Björkman (2003) Björkman and Ahrne (2005) Björkman et al. (2008) Dalin et al. (2004) Pullin and Gilbert (1989) Pullin and Gilbert 1989 Mutikainen and Walls (1995) Mutikainen and Walls (1995) Mutikainen and Walls (1995) Mutikainen and Walls (1995)

Reference

92 P. Dalin et al.

4 Leaf Trichome Formation and Plant Resistance to Herbivory

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density of leaf trichomes can be expected in environments that are dry or cold, where UV-radiation is intense, and in areas where the risk of being damaged by herbivorous insects is high (Ehrlinger 1984; Løe et al. 2007).

4.2 Leaf Trichome Formation 4.2.1 Molecular Basis of Leaf Trichome Formation Trichomes are initiated in the epidermis of developing leaves: some epidermal cells will develop into trichome cells whereas surrounding cells will develop into regular epidermal cells. Present understanding of trichome formation is based on studies of a few model species. In particular, trichome formation in A. thaliana has been used as a model system to study various developmental and cellular mechanisms in plants. In Arabidopsis, trichomes are non-glandular, large single cells, surrounded by accessory epidermal cells on rosette leaves, stems, cauline leaves, and sepals. With mutagenesis screens, dozens of genes involved in trichome initiation, spacing, and shape have been identified in Arabidopsis (Marks 1997; Hülskamp and Schnittger 1998). Recent studies of A. thaliana and its close relatives have shown that variation in some of the same genes can explain differences in trichome formation in natural populations (Hauser et al. 2001; Kivimäki et al. 2007). Studies of the genetic basis of trichome initiation have identified genes that (a) control the entry into the trichome pathway and (b) control the spacing of initiation events. Loss-of-function mutations in A. thaliana have suggested that the genes GLABROUS1 (GL1) and TRANSPARENT TESTA GLABRA1 (TTG1) are important for both the initiation and spacing of leaf trichomes. GL1 encodes a mybtranscription factor that is expressed diffusively in developing leaves (Oppenheimer et al. 1991). TTG1 is expressed in most major plant organs, codes for a WD-40 repeat-containing protein, and is needed for trichome cell initiation in leaves, nonhair cell type (atrichoblast) formation in roots, anthocyanin biosynthesis in leaves, and formation of mucilage layer in germinating seeds (Koornneef 1981; Walker et al. 1999). For normal trichome initiation, the genes GLABROUS3 (GL3) and ENHANCER OF GLABRA3 (EGL3), which code for helix-loop-helix (bHLH) proteins, are also needed (Payne et al. 2000; Zhang et al. 2003). GL1 and TTG1 have the capacity to limit trichome initiation together with other genes, e.g., CAPRICE (CPC) and TRIPTYCHON (TRY) that are known to promote root hair cell fate (Szymanski et al. 2000; Schiefelbein 2003). The gene GLABROUS2 (GL2) quantitatively regulates the frequency of trichome initiation and is involved in determining trichome spacing and root hair development (Ohashi and Oka 2002), but is also involved in the control of seed oil accumulation (Shen et al. 2006). Thus, many genes involved in trichome initiation and spacing also regulate other important phenotypic traits, which may constrain the evolution of genes and traits. Although current understanding of the genetic basis of trichome initiation and spacing depends to a large degree on studies of mutants in Arabidopsis, some

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information is also available for other plant species. Trichome initiation in cotton seems to be regulated by a similar multimeric complex between MYB, bHLH and WD repeat proteins as in Arabidopsis (Wang et al. 2004). Sequence comparisons and functional analyses suggest that genes underlying trichome formation in Arabidopsis (and perhaps cotton) may have evolved from duplication and neo-functionalization of genes involved in anthocyanin and flavonoid production, whereas the genes involved in the formation of analogous multicellular trichomes in Nicotiana and Antirrhinum have a different origin (Serna and Martin 2006). Thus, trichome production appears to have evolved several times suggesting convergent adaptive evolution.

4.2.2 Genetic Basis of Natural Variation in Trichome Production Both trichome production as such (trichome production vs. glabrousness) and the type of trichomes produced (glandular vs. non-glandular) may vary within species. Many plant species are polymorphic for trichome production with trichomeproducing and glabrous morphs. In several species, the frequency of the trichomeproducing morph varies geographically, among and within habitats (Björkman and Anderson 1990; Westerberg and Saura 1992; Barnes and Han 1993; St Hilaire and Graves 1999; Arroyo-Garc´ıa et al. 2001; Morrison 2002; Kärkkäinen et al. 2004; Løe et al. 2007). Genetic analyses have shown that the inheritance of the glabrous morph is simple in many species (one gene with a recessive allele for glabrousness; e.g., Westerbergh and Saura 1992; Sharma and Waines 1994; Kärkkäinen ˚ and Agren 2002). In A. lyrata, association analysis of phenotypic variation and genotypic variation in candidate genes revealed that the polymorphism in trichome production was caused by variation in GLABROUS1 (Kivimäki et al. 2007), and sequence analysis suggested that several independent mutations in the same gene cause glabrousness in different natural populations of A. lyrata and A. thaliana (Kivimäki et al. 2007; Hauser et al. 2001). Polymorphism in trichome morphology (glandular vs. non-glandular trichomes) can also be inherited in a simple Mendelian fashion, as documented for Datura wrightii (van Dam et al. 1999). The number of trichomes produced and trichome density vary genetically within several species. Quantitative genetic studies have indicated ample genetic vari˚ ation for trichome number and trichome density (Agren and Schemske 1994; Mauricio and Rausher 1997; Roy et al. 1999; Clauss et al. 2006). QTL-studies have been conducted to reveal the genetic basis of among-population differences in trichome formation. In A. thaliana, a QTL-analysis of a cross between two accessions identified a major QTL for trichome number (REDUCED TRICHOME NUMBER, RTN) that explained 70% of the variation in number of trichomes produced (Larkin et al. 1996). More recent studies of crosses among other accessions of A. thaliana detected additional QTLs (altogether nine QTLs in four different crosses, most of which were shared among populations; Symonds et al. 2005). Preliminary results from QTL-crosses and within-population studies in A. lyrata suggest that a limited


95

number of loci contribute to variation in the number of trichomes produced, and that some genes segregate in several populations also in this species (Kärkkäinen et al. unpublished data).

4.3 Induced Trichome Production 4.3.1 Damage-Induced Trichome Production Both artificial wounding and damage by herbivores can induce an increase in trichome density, and induction of trichome production has been observed in both annual and perennial plants (Table 4.1). The magnitude of the reported increase in trichome density is typically between 25% and 100%, but in some cases as large as 500%–1000% (Table 4.1). The responses often involve changes in trichome density expressed within days or weeks (Baur et al. 1991; Agrawal 1999, 2000; Rautio et al. 2002; Dalin and Björkman 2003). In some woody perennials, the response is delayed and not observed until the year following initial attack (Zvereva et al. 1998; Valkama et al. 2005). Damage can also induce a change in the relative proportions of glandular and non-glandular trichomes (Rautio et al. 2002). Abiotic stress, such as drought and UV-radiation, may influence trichome formation (Nagata et al. 1999; Höglund and Larsson 2005), and abiotic conditions may modify damage-induced responses in trichome density. For example, Björkman et al. (2008) showed that the increase in trichome production induced by leaf beetle herbivory in the willow Salix cinerea is stronger in the shade than under direct sunlight, suggesting that the plants invested more resources into trichome defense when growing in the shade. The results from Table 4.1 also show that damage-induced increases in trichome density tend to be stronger under field conditions than in the lab. All five studies, in which plants increased the density of leaf trichomes by more than 200% following damage, were conducted in the field. However, empirical data are still few, and additional studies of how abiotic conditions affect damage-induced responses in trichome formation are clearly needed. Damage has not been observed to induce trichome production in all studies (Table 4.1). Only about half of the 15 independent studies listed in Table 4.1, in which the experimental treatment consisted of artificial wounding (e.g., clipping of leaves) reported an increased trichome density. Also, the effect of damage on trichome production may vary depending on the identity of the herbivore; not all herbivorous insects induce a change in trichome formation (Traw and Dawson 2002a, b). Artificial wounding differs from the damage caused by the feeding of insects in several ways, which may reduce the chances of detecting induced responses (Hartley and Lawton 1987; Cipollini et al. 2003). Moreover, the feeding patterns of insects vary, especially among feeding guilds (e.g., chewers, miners and borers), which may affect how plants respond to damage. Further studies are therefore needed to examine the extent to which responses vary with damage type, and whether plants may benefit from responding differently to different kinds of damage.

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4.3.2 Hormonal Regulation of Induced Trichome Production Recent work on Arabidopsis indicates that several regulatory networks may influence damage-induced increases in trichome production. Jasmonic acid regulates the systemic expression of chemical defenses (Karban and Baldwin 1997; Schaller and Stintzi this volume), and herbivore damage as well as artificial wounding cause rapid increases in jasmonic acid (Bostock 1999; Reymond et al. 2000). In A. thaliana, artificial damage, but also application of jasmonic acid and application of giberellin increases trichome production in new leaves (Traw and Bergelson 2003). Application of salicylic acid, on the other hand, reduces trichome production and inhibits the response to jasmonic acid, which is consistent with negative cross-talk between the jasmonate- and salicylate-dependent pathways in Arabidopsis (Traw and Bergelson 2003). Concentrations of salicylic acid typically increase in response to infection by biotrophic pathogens (Gaffney et al. 1993; Ryals et al. 1994), but may also increase in response to damage caused by some herbivores (Stotz et al. 2002; van Poecke and Dicke 2002). This suggests that induction of increased trichome production will be affected by interactions with both herbivores and pathogens, and should vary depending on the identity of the herbivore.

4.4 Leaf Trichomes and Plant Resistance 4.4.1 Effects on Herbivore Behavior and Performance Trichomes influence insect oviposition and/or feeding in a wide range of insects and other herbivores (Levin 1973). Non-glandular trichomes mainly function as a structural defense against small herbivores. They interfere with the movement of insects and other small arthropods over the plant surface and make it more difficult for insects to access the leaf epidermis underneath for feeding (Southwood 1986). Trichomes are often composed of cellulose and other substances that constitute low nutritional value for the insects (Levin 1973). Insects that need to feed through the trichomes before accessing the leaf epidermis might therefore gain less weight and, ultimately, show increased mortality. Glandular trichomes can be viewed as a combination of a structural and chemical defense. This is because the glandular trichomes release secondary metabolites that can be poisonous or repellent to herbivorous organisms. Stinging trichomes, such as those produced by stinging nettles (Urtica dioica), may deter even large herbivores (Pollard and Briggs 1984). The presence and density of leaf trichomes can influence both host-plant selection behavior and performance (i.e., growth, survival, and fecundity) of herbivorous insects, but can be considered a relatively soft ‘weapon’ in plant defense compared with many other traits that are lethal to insects. It has been argued that weak defense traits might influence a wide range of herbivorous insects because they are less likely to result in counter-adaptations in insects (Feeny 1976). For example, there are a number of studies suggesting that leaf trichomes reduce the feeding of both generalist and specialist insects (Traw and Dawson 2002a; Agrawal 1999, 2004). Agrawal (1999) showed that two lepidopteran species, one specialist and


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one generalist, were both negatively influenced by previous damage on wild radish plants, which was correlated with an increased density and number of trichomes on the leaves. Agrawal (2004) also showed that several insect feeding guilds, both leaf chewers and leaf miners, were negatively affected by an increased density of leaf trichomes on milkweed (Asclepias syriaca). Thus, non-glandular trichomes might influence the feeding by a wide range of insects and therefore reduce total damage caused by herbivores. Insects may evolve physiological or behavioral traits that allow them to cope with structural plant defenses. For example, several mirid bugs (Heteroptera: Miridae) have special structures on their legs, which facilitate movement across trichomecovered plant surfaces (Southwood 1986; van Dam and Hare 1998). The leaf beetle Phratora vulgatissima has been observed to remove non-glandular trichomes when feeding on leaves of the willow Salix viminalis (Dalin et al. 2004). This behavior was not observed when the same beetle species was feeding on S. cinerea, which produces shorter and broader trichomes that appeared more difficult for the larvae to handle (Dalin and Björkman 2003). The evolution of mouthparts strong enough to handle structural plant traits is thus one possible mechanism for insects to circumvent structural plant defenses such as leaf trichomes (Levin 1973; Raupp 1985).

4.4.2 Effects on Tritrophic Interactions Leaf trichomes do not only affect herbivores, but also their natural enemies. This may indirectly affect the intensity of damage caused by herbivores. In theory, the effect on the abundance and effectiveness of natural enemies may be neutral (no effect), negative or positive (Fig. 4.1). If we assume that herbivores are affected negatively by leaf trichomes (Fig 4.1; top graph), a neutral effect on the third trophic level will result in natural enemies having an additive effect on plant damage caused by herbivores and on plant fitness. A negative effect on the third trophic level will act antagonistically. Depending on the relative strength of the negative effects of leaf trichomes on herbivores and natural enemies the correlation between trichome density and damage may vary from negative to positive. Plant damage will be (i) negatively correlated with trichome density if the effect on natural enemies is weaker than that on herbivores (dotted line) (ii) uncorrelated with trichome density if the effect on the natural enemies and herbivores balance (solid line), or (iii) positively correlated with trichome density if the effect on natural enemies is stronger than that on the herbivores (dashed line). A positive effect of leaf trichomes on the third trophic level should result in a synergistic positive effect on plant fitness. From a plant protection perspective, this last scenario would be most desirable, but there seem to be few such examples. In most empirical studies, the abundance and effectiveness of natural enemies were found to be negatively correlated with the density of plant trichomes (Kauffman and Kennedy 1989; Farrar and Kennedy 1991; Nihoul 1993; Gange 1995; Heinz and Zalom 1996; Romeis et al. 1998, 1999; Krips et al. 1999; Rosenheim et al. 1999; Lovinger et al. 2000; Fordyce and Agrawal 2001; Stavrinides and Skirvin 2003; Mulatu et al. 2006; Olson and Andow 2006). The effectiveness of both predators

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Herbivore performance or density without natural enemies Plant trichome density Neutral

Negative

Positive

Effect on natural enemies

Additive

Antagonistic

Synergistic

Plant damage

Fig. 4.1 Simplified graphical presentation of how the effect of leaf trichome density on the abundance or effectiveness of predators and parasitoids of herbivores may influence the relationship between trichome density and plant damage caused by herbivores (bottom row of graphs). The natural enemies may not be affected, or may be negatively or positively affected by plant trichome density (middle row of graphs). A basic assumption is that plant trichome density negatively affects herbivore performance or density (top graph), i.e., trichomes have the potential to function as a plant resistance trait (cf. Hare 1992)

(in 9 out of 13 reviewed studies) and parasitoids (in 12 out of 14 reviewed studies) is affected negatively because movement is inhibited and searching time is prolonged. Both non-glandular and glandular plant trichomes can have these effects. Glandular trichomes, in addition, affect natural enemies negatively by (a) the release of repellent or toxic compounds and (b) being sticky and hindering movements. However, the effect of the sticky substances produced by glandular trichomes may sometimes be of little importance in the field as the adhesive effect of the trichome exudates can be negatively affected by dust, wind, and rain (Obrycki and Tauber 1984; Romeis et al. 1999). In a few cases, the abundance or effectiveness of natural enemies were found to be positively correlated with the density of plant trichomes. For example, Styrsky et al. (2006) recently showed that fire ant predation of herbivores on soybean was higher on isolines of soybean with high trichome density. The suggested mechanism was a functional response by the ants to a higher abundance of caterpillars on pubescent plants. Apple trees producing high density of trichomes had a higher abundance of predatory mites than apple trees with a low density of trichomes, and it has been suggested that this was because pubescent plants capture more pollen and fungal spores that serve as alternative food for the predators (Roda et al. 2003). Lucas and Brodeur (1999) showed that within potato plants, an aphidophagous midge was more abundant on leaves with a high trichome density. The authors


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suggested that both a decreased risk of intraguild predation by e.g., coccinelids and a higher density of food (i.e., aphids) on leaves with a high trichome density may have contributed to this pattern. Finally, leaf trichome exudates may serve as extrafloral nectar for a scelonid egg parasitoid of squash bugs (Olson and Nechols 1995). Adult females of the parasitoid lived longer when they had access to squash leaves than when only provided with water, and this increase in longevity was enough to allow maximum realized fecundity. In even fewer cases, the abundance or effectiveness of parasitoids and predators were found not to be correlated with leaf trichome density (e.g., Sutterlin and van Lenteren 1997; Björkman and Ahrné 2005; see also reviews by Kennedy 2003; Simmons and Gurr 2005), possibly because of a reluctance to report ‘negative’ data. The density of prey or hosts can affect the impact of leaf trichomes on predators and parasitoids, but relationships appear system-specific. In some studies, an effect of trichomes on natural enemies was detectable only at high prey/host densities (e.g., Sutterlin and van Lenteren 2000) while in other studies such an effect was observed only at low densities (Krips et al. 1999).

4.4.3 Adaptive Significance of Induced Trichome Production in Plants The adaptive significance of damage-induced increases in trichome production should depend on the benefits and costs associated with this change in phenotype. Plants induced to increase leaf trichome density often receive less damage by insects than non-induced plants (Baur et al. 1991; Agrawal 1999; Dalin and Björkman 2003). This suggests that plants should benefit from induced trichome production as long as the fitness costs associated with increased trichome production do not exceed the benefits of reduced damage. Agrawal (1999) showed that induced wild radish plants (Raphanus raphanistrum and Raphanus sativus) received less damage by a variety of insects, and produced more seeds and fruits than noninduced plants. Wild radish responded to herbivory by producing new leaves with increased trichome density, but also with a systemic increase in the concentrations of secondary metabolites (Agrawal 1999). Thus, although this study clearly shows that the plants benefited from the induced responses, additional studies are needed to distinguish the effects of increased trichome production on fitness from those of other induced differences in plant quality in this and other species. Apart from reducing overall damage, induced trichome production may divert damage away from the most valuable parts of the plant and cause insects to feed on other, less valuable leaves, or other non-induced plant individuals nearby. Because plants cannot change the density of trichomes on already existing leaves, an induced increase in trichome production can only be expressed in leaves developing during or subsequent to attack (Traw and Bergelson 2003). Induced increases in trichome production can however be relatively rapid and expressed in new leaves within days or weeks after the initial attack (Baur et al. 1991; Agrawal 1999; Dalin and Björkman 2003). A similarly rapid reduction in trichome production in new leaves

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when damage ceases indicates that trichomes are costly to produce (Björkman et al. 2008). Additional evidence for costs of trichome production is provided by examples of trade-offs between trichome density and other putative defense traits, such as thorns (Björkman and Anderson 1990). A damage-induced increase in trichome production can lead to variation in trichome density within plants with older, basal leaves having lower trichome density than younger, induced apical leaves. Larvae of the leaf beetle P. vulgatissima showed a more dispersed feeding behavior among induced willow leaves with high trichome density at the top of shoots, indicating that the insects were searching for more optimal food within the branches (Dalin and Björkman 2003). Such damage-induced within-branch variation in trichome density may reflect developmental constraints, but may also be advantageous since damage to old basal leaves is likely to be less detrimental to plant fitness than damage to the young apical leaves (cf. Feeny 1976).

4.5 Perspectives Several plant species respond to herbivory by producing leaves with increased density and number of trichomes. The apparent benefit of this response is to reduce further damage by making it more difficult for insects to feed on the leaves. However, the magnitude of induced trichome production may vary with habitat conditions, and increased trichome density may affect predators and parasitoids negatively, which could reduce the effect on leaf damage. Moreover, trichome production may also affect tolerance to drought and other forms of abiotic stress. Taken together, this suggests that an analysis of the adaptive significance of induced trichome production requires a multidimensional view of the realized fitness (see also chapter by Steppuhn and Baldwin this volume). The effects of induced trichome production on interactions with herbivores should not be examined in isolation, but has to be considered in the context of effects on tritrophic interactions, and the extent to which fitness costs and benefits vary with abiotic conditions in natural populations. More information is also needed on the genetic and hormonal basis of induced trichome production. Such knowledge could be important for the development of sustainable pest control methods, using induced plant responses as a tool to prevent insect pest damage in agricultural systems.

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Chapter 5

Resistance at the Plant Cuticle Caroline Muller ¨

Resistance against herbivores could potentially start at the plant cuticle, the first contact zone between an approaching organism and the plant. Due to its physicomechanical and chemical properties the cuticle comprises several important physiological and ecological functions. Chemical compounds of or on the cuticle can directly act deterrent or even toxic to herbivores. The physico-mechanical cuticle characteristics influence locomotion and attachment of herbivorous insects but also of antagonists, thereby affecting direct and indirect constitutive defense properties. Little is known on induced resistance with regard to the plant cuticle. Induced direct defense could act through a production of waxes or biosynthesis of secondary metabolites that are deposited at the plant cuticle after herbivore attack or which are induced by compounds of oviduct secretion or footsteps. Furthermore, the specific cuticle properties influence the sorption of compounds originating from herbivorous species that could directly inhibit conspecifics, or affect the searching behavior of antagonists. An indirect induced defense can also be mediated by similarities between cuticular compound profiles between plants and herbivores that influence the palatability by predators. Finally, optical properties can change due to herbivory and affect the host finding behavior of predators or parasitoids. The deposition and behavior of agrochemicals used to increase crop resistance is highly determined by various cuticle characteristics. Different aspects of the role of the plant cuticle in resistance are outlined in this chapter, as well as potential future studies in this field.

5.1 Introduction The plant cuticle secludes all above-ground plant tissue from the environment and forms an important protection barrier against water loss by transpiration but also against biotic harms from outside. For any approaching organism this outer plant

C. Müller Department of Chemical Ecology, University of Bielefeld, D-33615 Bielefeld, Germany e-mail: [email protected]


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surface thus builds the first contact zone. Before herbivorous insects can damage the plant tissue, the cuticle can highly efficiently inhibit attachment, locomotion, feeding, or oviposition. Thereby it can be generally prevented that (not-adapted) insects will come into contact with diverse plant primary and secondary metabolites, and that damage could activate enzyme activities, thereby evoking induced defense responses of the plant. The plant cuticle is well characterized by its physico-chemical and morphological features. It forms a continuous translucent film of polymeric lipids and soluble waxes covering all aerial primary tissues of higher plants which is only interrupted by stomata. The cuticle consists of three main parts that are deposited at the outermost extracellular matrix of the epidermis: the cuticle layer forms the basis that embeds the cuticle proper which is covered by epicuticular waxes. The biopolyester cutin is composed of ␻-hydroxy and hydroxyepoxy fatty acids and offers a densely networked structural support. The cuticular waxes form a complex mixture of very long-chain fatty acids and their derivatives, and additionally often various cyclic compounds such as pentacyclic triterpenoids (Riederer and Markstädter 1996; Müller and Riederer 2005). The specific chemical composition leads to a specific ultrastructure of the cuticle, which can vary highly between species. Barthlott et al. (1998) classified a total of 23 wax types, which differ in thickness and in presence and shape of local wax projections, among other features. The physico-chemical properties of the cuticle influence features of the plant surface such as color, texture and presence of phytochemicals (volatile or non-volatile; Müller and Riederer 2005) that are important determinants for host plant acceptance by herbivorous insects (Powell et al. 1999). Generally, the cuticle will form a constitutive resistance barrier against herbivores that lack adaptations to a specific host plant surface. However, its fundamental role in plant-insect interactions is underrepresented in the scientific literature. Many more studies focused on the role of secondary plant compounds for insect acceptance or rejection (Schoonhoven et al. 2006) that are, however, often not present on the outer surface but only in inner plant tissue (Müller and Riederer 2005). Studies on resistance effects of the plant cuticle were mainly driven by investigations of crop pests (e.g., Eigenbrode and Shelton 1990; Stoner 1990; Cervantes et al. 2002). Comparisons between acceptance of plant genotypes or varieties with different levels of wax covers helped to elucidate the role of constitutive resistance parameters of plant individuals (Yang et al. 1991; Eigenbrode and Pillai 1998; Cervantes et al. 2002). Furthermore, indirect constitutive defense can be implemented at the plant cuticle, as the cuticle does not only determine features for herbivorous insects but also for predators or parasitoids, specifically by influencing their locomotion abilities (Eigenbrode 2004). The knowledge of the role of the plant cuticle in induced resistance on the other hand is extremely poor, and offers a comprehensive interesting field for future studies. This chapter will start with a description of the physico-chemical nature of the plant cuticle which is essential to explain features that determine the role of the cuticle in interactions with herbivores. In the following, constitutive resistance characteristics of the plant against herbivores will be highlighted which can be divided in direct resistance and indirect, the latter acting by influencing the efficiency of predators or parasitoids of the herbivores. The underlying mechanisms can be based

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on physical and/or chemical features of the cuticle. Few examples will be given where the plant cuticle has been found to be involved in induced resistance, either direct or indirect. As agrochemicals need to intrude the plant tissue through the cuticle and thereby might change the plant surface considerably, the impact of crop protection treatments on plant-insect interactions will be shortly discussed. The chapter will end with an outlook, suggesting some future studies that might be relevant to gain a deeper understanding on the role of the plant cuticle as a plant resistance factor.

5.2 Characteristics of the Cuticle 5.2.1 Physico-Chemical Features The plant cuticle forms a hydrophobic outer barrier of nearly all above-ground parts of terrestrial plants. Its thickness can vary from 0.01 to 200 ␮m (Nawrath 2006). The cuticle is a bilayered membrane. At first, the cuticle proper consisting of soluble and polymeric lipids is synthesized at the outside of the primary cell wall. During organ development, a zone of the primary cell wall and later of the secondary cell wall becomes impregnated with cutin, forming the cuticle layer beneath the cuticle proper which can vary in thickness (Jeffree 1996, 2006; Bargel et al. 2006). The cutin matrix (intracuticular) is composed predominantly of C16 ␻-hydroxy and unsaturated C18 hydroxyepoxy fatty acid monomers (Holloway 1982) and contains to a lesser extent also glycerol and phenolic compounds (Nawrath 2006). Furthermore, various cell wall polysaccharides are present in the cuticle layer (Holloway 1982). Cuticular waxes are typically comprised of a complex mixture of aliphatic long-chain hydrocarbons together with cyclic compounds. The major classes of cuticular waxes are n-alkanes (C17 –C35 ), smaller portions of iso- and anteiso homologues, primary alcohols (C22 –C32 ), fatty acids (C12 –C32 ), aldehydes (C22 –C36 ), secondary alcohols (C21 –C33 ) ␤-diketones (C27 –C35 ), and n-alkyl esters (C36 –C72 ) (Walton 1990; Jeffree 2006). Additionally, various triterpenoids can be found, most of them being pentacyclic triterpenoids (Walton 1990; Jetter et al. 2006). For a thorough review of the composition of plant cuticular waxes see Jetter et al. (2006). The intracuticular waxes of the cuticle proper mainly form well-packed subdomains, while the epicuticular waxes are deposited as film or form crystals (Müller and Riederer 2005). Intra- and epicuticular waxes can be sampled separately (Ensikat et al. 2000) and have been shown to have distinct qualitative and quantitative compositions of wax compounds (Jetter et al. 2000, 2006). After removal or abrasion of waxes, a constant regeneration usually follows (Neinhuis et al. 2001). While wax films appear to be ubiquitous, thick wax layers are rare. The crystals can vary from one to tens of micrometers in size and their shape is determined by the qualitative and quantitative composition of wax compounds (Bargel et al. 2006; Jeffree 2006). Among the crystalloids, platelets, tubules, stomatal wax chimneys, rodlets, needles, ribbons and others can be found (Jeffree 1986; Barthlott et al. 1998), which determine the microrelief of the plant surface.

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The microrelief influences several features of the plant surface as e.g., the wettability. While smooth surfaces, lacking wax crystals, are more or less wettable, rough surfaces offer permanent water repellency (Neinhuis and Barthlott 1998). The roughness can be caused by different microstructures such as wax crystals and cuticular folds, but also by trichomes (Neinhuis and Barthlott 1997). A rough surface helps to keep the cuticle dry and clean, as mechanical adhesion of small particles is impaired and water droplets running off the surface take along particles (Barthlott and Neinhuis 1997). In the plant-insect literature, the different microreliefs are usually simplified and only the terms ‘glossy’ and ‘waxy’ are used. Glossy plants have usually a reduced complexity of the microstructure and reduced amounts of epicuticular lipids in comparison to normal wax phenotypes (Eigenbrode and Espelie 1995). Generally, glossy plants allow an easier attachment for insects than normal or extremely waxy surfaces (Eigenbrode and Pillai 1998; Gorb and Gorb 2002; Eigenbrode 2004) but these features can also differ depending on the specific plant-insect system. Furthermore, the microrelief of the plant cuticle strongly influences its optical properties (Pfündel et al. 2006). On the one hand radiation is absorbed at the plant surface, but on the other hand photons are also redirected at the transition of phases with different refractive indices causing specular reflectance (Vogelmann 1993; Barnes and Cardoso-Vilhena 1996). Electronic absorption of radiation depends on the presence of linear or cyclic compounds including several double bonds (Cockell and Knowland 1999) such as phenolics, hydroxycinnamic acids, colorless flavonoids or anthocyanins (Krauss et al. 1997; Olsson et al. 1998; Harborne and Williams 2000; Markstädter et al. 2001). Their primary role is the protection against damage caused by ultraviolet radiation, but their qualitative and quantitative occurrence in the cuticle also determines its optical behavior. The specular reflectance of the radiation depends on the angle of incident radiation and on the viewing angle of the observer, e.g., an approaching insect (Woolley 1971). Upper surfaces of glabrous leaves exhibit reflectance values in the UV-A spectral range (315–400 nm) between four and 10% (Caldwell 1968; Bullas-Appleton et al. 2004), while glaucous leaf surfaces with a blue–green appearance reflect radiation up to 25% (Pfündel et al. 2006). In several succulent plants with powdery epicuticular waxes a reflectance of UV up to 80%, and of visible radiation (400–700 nm) up to 70% can be reached on the upper leaf surfaces (Mulroy 1979). Furthermore, trichomes can influence the reflectance of radiation to highly variable degrees (Ehleringer and Björkman 1978; Robberecht et al. 1980; Baldini et al. 1997). Combination of absorption and specific wavelength reflectance properties can finally influence the colored appearance of the cuticle which can be used as cue for host plant finding of herbivorous insects (Prokopy and Owens 1983; Prokopy et al. 1983; Bullas-Appleton et al. 2004).

5.2.2 Further Chemical Characteristics Next to the typical wax compounds or in combination with these, other plant metabolites whose presence might be important in plant-insect interactions were


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identified in the cuticle over the last decades. Wax fatty acids can esterify with phenylpropanoids (cinnamyl alcohol), diterpenes (phytol) (Griffiths et al. 1999), benzyl and phenylethyl alcohol (Gülz and Marner 1986) or alkyl benzoates (Gülz et al. 1987). Very long-chain phenylpropyl and phenylbutyl esters have been found in needle cuticles of Taxus baccata L. (Taxaceae) (Jetter et al. 2002). Tocopherols have been reported in the waxes of a number of species (Griffiths et al. 2000; Gülz et al. 1992). Soluble carbohydrates and amino acids can leak to the leaf surface (Fiala et al. 1990; Soldaat et al. 1996; Derridj et al. 1996a; Leveau 2004) and offer information about the quality of the plant as a food source available to insects (Derridj et al. 1996b). However, the leaking of carbohydrates onto the surface is size-limited, since large carbohydrates will not be able to pass the cuticle (Popp et al. 2005). Compounds located in trichomes can be exuded or released onto the plant surface when the cuticular sac of the trichomes matures. Diterpenoids, sesquiterpenes and flavonoids have been reported to be deposited on the leaf surface via this way (Severson et al. 1984; Talley et al. 2002; Roda et al. 2003; Bargel et al. 2004). But flavonoids and other phenolic compounds are also found on the surface of diverse higher plant families independent of trichomes (Wollenweber et al. 2004, 2005a, b). In ferns, flavonoids can form thick farinose layers on the fronds (Wollenweber 1989; Roitman et al. 1993). Various secondary metabolites, which can be important infochemicals in plantinsect interactions, were described to be present in leaf surface extracts, such as pyrrolicidine alkaloids in Asteraceae (Vrieling and Derridj 2003), glucosinolates in Brassicaceae (Städler 1992; Hopkins et al. 1997; Griffiths et al. 2001; van Loon et al. 1992), terpenoids (Hubbell et al. 1984) but also phytosterols (Holloway 1971; Hubbell et al. 1984; Avato et al. 1990; Shepherd et al. 1999a). However, these findings are discussed controversially, specifically when the metabolites are rather non volatile, polar and/or charged (Müller and Riederer 2005). Surface extracts prepared with organic solvents, used in several of the mentioned studies for isolation of ‘cuticle compounds’, always hold the risk that compounds are eluted from underlying plant tissue and do not originate from the cuticle (Riederer and Markstädter 1996; Jetter et al. 2000, 2006). Soldaat et al. (1996) did not find pyrrolicidine alkaloids in surfaces of Senecio (Asteraceae) and the only alkaloids that have been confirmed as cuticle constituents with more sophisticated methods are these of fruits of Papaver (Papaveraceae) (Jetter and Riederer 1996). Also, the often reported presence of glucosinolates on the plant cuticle is not certain (Reifenrath et al. 2005). Keeping plants in darkness before solvent extraction lowers the risk of dissolving compounds from the inner leaf through stomata (Reifenrath et al. 2005). Preferably, extractions with organic solvents should be avoided altogether and replaced by different adhesion methods using frozen water or glycerol (Ensikat et al. 2000; Riedel et al. 2003), cellulose acetate (Müller and Hilker 2001), or gum arabic (Jetter et al. 2000; Riedel et al. 2003; Reifenrath et al. 2005), to mechanically detach the epicuticular waxes from the plant surface for chemical analyses of compounds present therein (Jetter et al. 2006; Müller 2006). Lipophilic uncharged organic chemicals, specifically volatiles, can easily accumulate in the cuticle and be sorbed onto plant surfaces whereby the cuticle can

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function as fixative (Müller and Riederer 2005). Volatile compounds could reach the surface via transport from the inner leaf through the cuticle (depending on the molecule’s characteristics) or by sorption from the environment. These volatiles can function as infochemicals for insects (Lambdon et al. 1998).

5.2.3 Variation of the Plant Cuticle The plant cuticle may vary in microstructure, thickness and chemical composition within an individual due to different endogenous and exogenous factors. Adaxial and abaxial surfaces can differ dramatically in chemical composition and ultrastructure (Netting and von Wettstein 1973; Holloway et al. 1977; Baum et al. 1980) which in turn influences feeding and oviposition by herbivores (Müller 1999; Müller and Hilker 2001). Age, developmental stage and organ are important endogenous determinants of leaf and fruit cuticle characteristics (Freeman et al. 1979a, b; Atkin and Hamilton 1982; Edwards 1982; Riederer and Schönherr 1988; Bianchi et al. 1989; Neinhuis and Barthlott 1998; Jetter and Schäffer 2001; Ficke et al. 2004). During senescence, reflectance and scattering patterns change as well, influencing the optical properties of the leaf surface (Major et al. 1993), and thereby offering visual information about the plant quality. Climate conditions in a certain habitat such as season (Gülz and Boor 1992; Neinhuis and Barthlott 1998; Jenks et al. 2002), or temperature (Gülz and Boor 1992; Riederer and Schreiber 2001) are important exogenous factors affecting cuticle characteristics, as well as erosion by wind and rain (Baker 1974; Baker and Hunt 1986; Rogge et al. 1993), or certain gases (Percy et al. 2002). Endogenous and exogenous factors might interact, as for example the presence of essential oils together with high temperature may lead to melting of wax platelets (Gülz and Boor 1992). Changes of the plant cuticle can be also caused by visiting insects (see Section 5.4) or by pathogens.

5.3 Constitutive Resistance 5.3.1 Physico-Mechanical Direct Defense Due to the specific morphology of the surface epicuticular waxes, the plant cuticle forms a constitutive resistance barrier against many visiting insects. The protection of flowers from undesired visitors has already early been described by Kerner (1879), and is realized by ‘antirobbing’ devices that cover the pedicles. Leaves of Ilex aquifolium (Aquifoliaceae) are so smooth that many insects cannot grasp them (Jolivet 1988). Attachment and locomotion of insects’ eggs and tarsae, respectively, can be strongly inhibited depending on the amount and structure of epicuticular waxes (Peter and Shanower 2001). The movement and feeding of neonate larvae in particular is affected by the specific constitution of the cuticle


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(Zalucki et al. 2002). The less complex epicuticular microstructure of glossy surfaces can lead to ten times lower survival of Plutella xylostella L. larvae (Lepidoptera: Plutellidae) compared to survival on waxy genotypes (Eigenbrode and Shelton 1990). Initiation of feeding and walking speed of these larvae are reduced on glossy as compared to waxy genotypes. Sorghum (Poaceae) genotypes with a smooth amorphous wax layer and sparse wax crystals are more resistant to the sorghum shoot fly Atherigona soccata (Rondani) (Diptera: Muscidae) than other genotypes (Nwanze et al. 1992). Thus, glossy surfaces can provide an important resistance factor against herbivores (Stoner 1990). Other resistance mechanisms might involve pleiotropic effects of the glossy trait on further plant characteristics, such as e.g., water stress (Cole and Riggal 1992; Rutledge and Eigenbrode 2003). Overall, agricultural crop species with glossy surfaces and reduced wax blooms are more often resistant to insect pests within the reported occurrences than species or varieties with waxy surfaces, whereby the resistance was mainly investigated against neonate caterpillars or smaller insects such as aphids or thrips (Eigenbrode 2004). The increased ability of these insects to walk on waxy surfaces might have evolved as part of the host plant specialization process. On the other hand, several insects have trouble adhering properly to waxy surfaces, as e.g., flea beetles (Coleoptera: Chrysomelidae) on waxy cabbage genotypes (Bodnaryk 1992). This results in reduced feeding on such plants. Waxblooms also prevent attachment of the mustard leaf beetle, Phaedon cochleariae (F.) (Coleoptera: Chrysomelidae), whose traction is much better on glossy leaf surfaces (Stork 1980). Leaves can change their microstructure when maturing (Edwards 1982), turning from a more diverse array of epicuticular waxes to a glossy type, as e.g., in various eucalypt species. The young eucalypt leaves are more resistant to attack by the tortoise beetle Paropsis charybdis St˚al (Coleoptera: Chrysomelidae) (Edwards 1982) and to oviposition and settling by the psyllid Ctenarytaina spatulata Taylor (Homoptera: Psyllidae; Brennan et al. 2001), whereas they are more susceptible to the autumn gum moth, Mnesampela privata (Guenée) (Lepidoptera: Geometridae) (Steinbauer et al. 2004). Normal or waxy surfaces of leaves and fruits can receive more (Steinbauer et al. 2004) or fewer eggs than glossy phenotypes (Neuenschwander et al. 1985; Uematsu and Sakanoshita 1989; Blua et al. 1995), the latter leading to an increased pest resistance. Different visual appearances of glossy and waxy phenotypes may contribute to acceptance by insects that use leaf color to distinguish between host plants (Prokopy et al. 1983; see Section 5.2.1). Mechanical removal of plant epicuticular waxes often led to a higher susceptibility to herbivores, shown e.g., for several psyllids (Brennan and Weinbaum 2001), chrysomelid beetles (Edwards 1982; Reifenrath et al. 2005; Müller 2006), and moths (Hagley et al. 1980; Uematsu and Sakanoshita 1989). Cellulose acetatestripped oat leaves were penetrated earlier by aphids than intact oat leaves (Powell et al. 1999). In contrast, monophagous herbivores might need intact epicuticular layers for host detection (Müller and Hilker 2001). The abundance of normal and reduced wax blooms can finally have effects on arthropod diversity (Chang et al. 2004b).

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5.3.2 Chemical Direct Defense The chemical composition of the cuticular lipids can cause resistance against several insect herbivores (Eigenbrode and Espelie 1995) through perception of stimuli by highly adapted sensory structures (Städler 1984, 1986) followed by avoidance behavior. The antixenotic activity of hexane and chloroform surface extracts of some grasses is higher than that of others against the Hessian fly (Diptera: Cecidomyiidae; Foster and Harris 1992; Cervantes et al. 2002). Applying epicuticular waxes of resistant on susceptible Rhododendron genotypes (Ericaceae) caused a reduction of feeding and oviposition of Stephanitis pyrioides Scott (Heteroptera: Tingidae; Chappell and Robacker 2006). Meridic diet containing cuticular lipid extracts from foliage of four maize genotypes (Poaceae) led to reduced growth of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae; Yang et al. 1991). Cultivars of Medicago sativa that contain 50% more wax esters are more resistant to the aphid Therioaphis maculata (Buckton) (Homoptera: Aphididae; Bergman et al. 1991). Deterrent effects of surfaces waxes of Sorghum seedlings against Locusta migratoria L. (Orthoptera: Acrididae) are attributable to p-hydroxybenzaldehyde, n-alkane, and ester fractions (Woodhead 1983). Addition of n-alkane-1-ols, specifically C24 and C25 alcohols, to cabbage wax components deposited on a glass slide reduced the time biting, and increased the time walking of neonate P. xylostella (Eigenbrode and Pillai 1998). The 3-O-fatty acid ester derivatives (C12 –C18 ) of two pentacyclic triterpenic acids, ursolic acid, and oleanolic acid, together with the parent acids, were found to exhibit potent antifeedant activities against the agricultural pest Spodoptera litura F. (Mallavadhani et al. 2003). The triterpenols ␣- and ␤-amyrin inhibit feeding of L. migratoria and reduce acceptance behavior of P. xylostella (Eigenbrode and Espelie 1995). Cultivars of Rubus idaeus L. (Rosaceae) with higher levels of cycloartenyl esters, ␣-amyryl esters and other compounds present in the cuticular waxes are more resistant to the aphid Amphorophora idaei Börner (Homoptera: Aphididae; Shepherd et al. 1999b). Sesquiterpenes located in trichomes of Lycopersicon hirsutum f. hirsutum (Solanaceae) are toxic to neonate larvae of Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae; Carter et al. 1989). Adults of the Colorado potato beetle abandon certain wild Solanum species by falling to the ground after a few minutes of contact with the plant surface. While this behavior is induced by trichomes, non-volatile fractions of leaf surface extracts were identified as phagodeterrents against the beetle (Yencho et al. 1994; Pelletier and Dutheil 2006). However, these extracts might contain also other compounds than cuticular waxes (see also Section 5.2.2). Toxic or deterrent secondary compounds can be concentrated to higher levels in glossy as compared to waxy surfaces and thus increase plant resistance (Cole and Riggal 1992). On the other hand, epicuticular waxes might also cover secondary compounds that act as feeding or oviposition stimulants and thereby increase plant resistance features (Reifenrath et al. 2005).


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5.3.3 Constitutive Indirect Defense An indirect constitutive defense of the plant against herbivorous insects can be realized by impacting the locomotion and attachment abilities of predators or parasitoids on the cuticle. Differences of leaf micro morphology and chemistry lead to different predation efficiencies of carnivores. The efficiency of predators increased several-fold on glossy genotypes compared to waxy (Eigenbrode and Espelie 1995; Eigenbrode et al. 1999). Specifically, coccinnelid larvae and beetles often fall off plants, or forage aphids less efficiently on plants with a pronounced epicuticular wax bloom (Grevstad and Klepetka 1992; Shah 1982; Rutledge et al. 2003). Thus, glossy phenotypes improve effectiveness of some predators leading indirectly to a higher resistance against herbivores. Selection and breeding of such phenotypes has been used especially for management of cabbage pests (Eigenbrode 2004). Similar effects could be observed with parasitoids. Whereas Diaeratiella rapae (M’Intosh) wasps (Hymenoptera: Braconidae) foraged more slowly, groomed with a higher frequency, and fell off from leaves of cauliflower varieties with heavier wax blooms more frequently, their foraging efficacy on cabbage aphids improved highly when epicuticular waxes were removed (Gentry and Barbosa 2006). Wasps of Aphidius ervi Haliday (Hymenoptera: Aphidiidae) spent more time actively foraging on pea plants with reduced wax loads, which may contribute to a higher parasitism rate of the pea aphid Acyrthosiphon pisum Harris (Homoptera: Aphidae) observed on those varieties (Chang et al. 2004a). However, predators and parasitoids do not seem to prefer plants with reduced wax blooms over waxy leaves, they just show higher success rates of predation and parasitation on these plants due to a much better attachment on glossy leaves (Eigenbrode and Kabalo 1999; Eigenbrode et al. 1999; Eigenbrode 2004). In contrast, several predator species can actually forage more efficiently on waxy surfaces (Eigenbrode 2004). A few syrphid species were shown to oviposit preferentially on plants with heavy epicuticular wax blooms, thereby increasing the plant resistance against herbivores (Rutledge et al. 2003). In some predator species, different developmental stages have contrasting abilities to cope with the surface structure of the plant. Parasitoids were also shown to differ in their ability to walk and forage on glossy versus waxy plant cuticles (Eigenbrode 2004). Further structures on the surface, such as trichomes, pronounced leaf veins, or smaller diameter stems or tendrils might help carnivorous insects to find a better grip to the plant (Kareiva and Sahakian 1990; Federle et al. 1997; Olson and Andow 2006). Finally, in ant-plant mutualistic relationships the physico-chemical features of epicuticular waxes play an important role for attachment abilities of specialized and non-specialized ant species. A stable association with an efficient ant species can increase the resistance of the plant against herbivores which are foraged by the ants. Different Macaranga plant-ant associations, and the involvement of the particular surface characteristics of the cuticle in these interactions have been studied in detail by Federle and coworkers (Federle et al. 1997, 2000; Markstädter et al. 2000; Federle and Bruening 2005).

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Whereas constitutive direct defense has been shown to be influenced by physico-mechanical characteristics of the cuticle (Section 5.3.1) and by the presence of certain chemical compounds on the surface (Section 5.3.2), indirect constitutive defense mechanisms are mainly driven by physico-mechanical properties.

5.4 Induced Resistance 5.4.1 Induced Direct Defense Plants can respond with a number of induced defense responses to herbivore feeding. In many plant species, changes in primary composition, as e.g., the C/N ratio (Ohgushi 2005), as well as changes of secondary metabolite concentrations in response to herbivory have been found that led to increased plant performance (e.g., Bennett and Wallsgrove 1994; Agrawal 1998; van Dam et al. 2001). Also, proteinase inhibitors can be induced by damage (Bergey et al. 1996; Koiwa et al. 1997; Ryan 2000; Tamayo et al. 2000), lowering the digestibility of the plant material for herbivores and thereby increasing plant resistance. The herbivores themselves show different levels of adaptation to overcome these plant defense responses, which can also be divided into constitutive and induced (Gatehouse 2002). While considerable knowledge has accumulated on mechanisms that lead to an induction of defense traits in the plant tissue after mechanical or herbivore damage (Kessler and Baldwin 2002; Arimura et al. 2005), relatively little is known on inducible effects on the plant cuticle. Direct effects on wax production were observed by Bystrom et al. (1968) after aphid punctures in Beta vulgaris L. (Chenopodiaceae). Infestation of leaves of Sorghum halepense (L.) (Poaceae) by Sipha flava (Forbes) (Homoptera: Aphididae) led to a reduction of crystalline epicuticular waxes compared to non-infested ones. However, infested leaves became even more susceptible to the aphid than intact plants (Gonzales et al. 2002). At the leaf surface, the density of trichomes can increase in response to herbivory (Agrawal et al. 2002; Traw and Dawson 2002; Valkama et al. 2005; Dalin et al. this volume). The induced trichomes can exude various metabolites such as e.g., flavonols (Roda et al. 2003), or other lipophilic compounds (Valkama et al. 2005) that change the surface chemistry of the plants. These responses can appear quite delayed in long-living species such as trees: the density of glandular trichomes and concentrations of several lipophilic compounds on the leaf surface increased in silver birch (Betula pendula Roth) defoliated during the previous summer. Effects were also visible in systemic branches and affected different herbivore guilds to various degrees (Valkama et al. 2005). Treatment of celery leaves (Apium graveolens, cv. secalinum, Apiaceae) with jasmonic acid or amino-acid conjugates of jasmonic acid stimulates the biosynthesis of a number of furanocoumarins which are at least in part deposited on the leaf surface (Stanjek et al. 1997). Whereas these act as oviposition stimulants for some specialists, they likely also have deterrent effects on generalist herbivores.


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Wounding by insects and the detection of insect elicitors originating from oral secretions by plants can lead to the release of systemin and other polypeptides that systemically activate plenty of defensive genes and signal transduction pathways, including the jasmonic acid signaling pathway (Bergey et al. 1996; Ryan 2000; Kessler and Baldwin 2002; Arimura et al. 2005). But even without any damage of the plant tissue, plants can build up resistance by responding to the mere touch of a crawling insect (Bown et al. 2002). Footsteps of crawling non-wounding insect larvae stimulated the synthesis of 4-aminobutyrate and superoxide within short time which might mediate rapid local resistance mechanisms (Bown et al. 2002). The superoxide production, leading to local cell death, might also play an important function in protection against infection by opportunistic pathogens at the site of the larval footprint damage (Hall et al. 2004). The response of the plant to insect crawling could be mimicked by suction applied with a micropipette (Bown et al. 2002), suggesting a purely physico-mechanical effect of larval pads on the plant surface. However, attachment pads on the tarsae of insects produce also fluid secretions that are needed for wet adhesion to the plant surface (Eisner and Aneshansley 2000; Gorb 1998; Federle et al. 2002; Vötsch et al. 2002; Federle and Bruening 2005). While the chemical composition of these fluids has only been studied in a small number of species (Attygalle et al. 2000; Betz 2003; Vötsch et al. 2002), they likely etch cuticular waxes. Etched wax crystals have been found that form an amorphous mass on adhesive pads influencing locomotion of insects (Gaume et al. 2004; Federle and Bruening 2005). These fluid secretions and their impact on epicuticular waxes could potentially be important stimuli for the plant to induce resistance responses. Another elicitor for early defense responses of plants may be present in oviduct secretions which are emitted during oviposition (Hilker and Meiners 2006). When eggs are only attached to the cuticle and leaves are not visibly damaged by oviposition it is unclear how potential elicitors could enter the plant tissue. Müller and Rosenberger (2006) showed that oviposition secretion can be sorbed in the plant cuticle in such species (i.e., in Lilioceris lilii L., Coleoptera: Chrysomelidae) and could even penetrate through stomata (in L. merdigera (L.)). This way signals may reach inner plant tissues allowing the plant to respond to insect attack with the induction of direct or indirect defense responses. Finally, sorption of compounds released from individual herbivores on the cuticle might affect conspecifics. The iridoid (epi)-chrysomelidial, which is the main component of the larval secretion of the leaf beetle P. cochleariae, was detectable in surface extracts of leaves on which larvae had fed (Rostás and Hilker 2002). The secretion has a deterrent effect on conspecific adults. Oviposition deterring pheromones have been reported in a number of species of different orders (Anderson 2002). Female tephritid flies mark the fruit with such pheromones after oviposition. As the fruits can only support a limited number of larvae, other females will avoid marked fruits as potential hosts for their offspring. The persistence of the pheromone varies from a few hours up to several days and depends on the nature of the pheromone and the physico-chemical properties of the cuticular surface (Müller and Riederer 2005).

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5.4.2 Induced Indirect Defense Cuticular lipids can hold important functions in multitrophic interactions, as their major constituents can be chemically very similar between cuticles of plants, herbivores, and their predators. The cuticular lipid components of larvae of Manduca sexta L. (Lepidoptera: Sphingidae) were shown to vary slightly depending on the surface composition of their specific host plants. This diet-related variation influences palatability by predatory ants (Espelie and Bernays 1989) and thus indirectly plant resistance features. The migratory grasshopper Melanopus sanguinipes (F.) (Orthoptera: Acrididae) incorporates n-alkanes of the plant cuticle in its integument (Blomquist and Jackson 1973) which is exploited for host recognition by some predators and parasitoids (Espelie et al. 1991). However, such kind of mimicry can also lead to perfect protection: larvae of Mechanitis polymnia (L.) (Lepidoptera: Nymphalidae) are defended against the predatory ant Camponotus crassus Mayr (Hymenoptera: Formicidae) by a chemical similarity between their cuticular lipids and those of the solanaceous host plant (Henrique et al. 2005). On other plant species, the larvae do not receive this chemical camouflage and experience higher predation rates. Plant cuticular hydrocarbons of fruits and seed coats of apples were detected in larvae and adults of codling moths, Cydia pomonella L. (Lepidoptera: Torticidae), and were furthermore identical between adult moths and their parasitoid Ascogaster quadridentata Wesmael (Hymenoptera: Braconidae). Even in the hyperparasitoid Perilampus fulvicornis Ashmead (Hymenoptera: Perilampidae) one cuticular lipid, pentacosene, was identical with its host (Espelie and Brown 1990). These relationships may have important implications for the biology of the members of the different trophic levels, for host acceptance and suitability. Indirectly, they again might affect plant resistance features. Feeding of a leaf mining herbivore was shown to lead to increased amounts of the triterpene squalene on the leaf surface of apples, which mediated host location of a parasitoid (Dutton et al. 2002). Induced wax production after aphid punctures that has been observed in B. vulgaris (Bystrom et al. 1968; see also Section 5.4.1) may also be used for host location by parasitoids. The volatility of compounds which are induced by feeding (Dicke et al. 1998; Kessler and Baldwin 2001; Mattiacci et al. 2001; Röse and Tumlinson 2004; Arimura et al. 2005) or oviposition (Meiners and Hilker 1997; Hilker et al. 2002; Colazza et al. 2004; Fatouros et al. 2005), and which are attractive to members of the third trophic level is certainly influenced by epicuticular lipids at the plant surface which thereby influence indirect plant resistance traits. The sorption of volatile organic compounds in the waxes (Welke et al. 1998) and the transport pathways through the cuticle (Riederer and Friedmann 2006) are highly determined by the lipophilicity of the compounds, by the saturation vapor pressure in the atmosphere above the cuticle (Riederer et al. 1995, 2002), and by the specific physicochemical characteristics of the plant cuticle (Müller and Riederer 2005). For studies on attraction of parasitoids by herbivore-induced volatiles it would be important to understand the kinetics of transport, and to know the concentrations of the key compounds, at the plant surface and in a distance.


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Herbivore infestation can also influence the optical appearance of the plant surface. Predators with good color perception will react to slight changes and predate more efficiently. For example, willow warblers were shown to respond to changes in UV spectral maxima which resulted from induction of flavonoids in the surface due to sawfly damage (Mäntylä et al. 2004). Endophytic feeding can lead to desiccation and discoloration, influencing chromatic and achromatic cues, mainly by changes of contrasts, for parasitoids searching for pupal hosts (Fischer et al. 2004). Also, polarized reflectance, determined by specular reflectance of the epicuticular waxes and surface particle scattering (Grant et al. 1993), may be changed by mining insects and influence host finding by carnivores.

5.5 Crop Protection The nature of the plant cuticle highly influences the deposition and subsequent behavior of pesticides, growth regulators, foliar nutrients, or other chemicals used for plant cultivation. For systemic crop protection, many agrochemicals are sprayed on the leaves where they first have to penetrate the plant cuticle whose waxy surface film and embedded cuticular waxes act as an important barrier. Organic non-electrolytes physically diffuse within and across the cuticle with varying mobility, depending on plant species and ontogenetic state, the solute size and shape, the temperature and the presence of accelerators (Buchholz 2006; Riederer and Friedmann 2006). Accelerators enhance the fluidity of cuticular waxes by acting as plasticizers (Schreiber 2006), thereby facilitating the subsequent penetration of lipophilic non-electrolytes (Riederer and Friedmann 2006). The plethora of compounds used as agrochemicals and as transport-enhancing chemicals via the cuticle has been reviewed from the plant perspective (Riederer and Schönherr 1990; Baur 1998; Zabkiewicz 2000). The specific nature of the cuticle also determines the persistence of the various agrochemicals on the plant surface (Riccio et al. 2006). However, little is known how changes of the morphological and physico-chemical characteristics of the cuticle after treatment with insecticides or herbicides (Taylor et al. 1981; Whitehouse et al. 1982; Gouret et al. 1993) act on herbivorous insects. Given that the cuticle plays an important role for host recognition, host acceptance, and also attachment abilities of insect legs and eggs, a tremendous effect on plant-insect interactions can be expected by changing plant surface features through application of agrochemicals. Herbicide treatment of Brassica spp. with S-ethyl dipropylthiocarbamate resulted in a reduction of epicuticular waxes (Eigenbrode and Shelton 1992; Justus et al. 2000). Treated plants turned more acceptable for oviposition by adult P. xylostella (Justus et al. 2000). The survival of neonate larvae was significantly reduced on these plants, however (Eigenbrode and Shelton 1992). Insecticide treatment not only kills the herbivores but may also negatively affect beneficial insects such as predators or parasitoids. Deltamethrin, used as insecticide, was shown to rapidly diffuse in oilseed rape cuticles. The parasitoid Diaeretiella rapae (M’Intosh) (Hymenoptera: Braconidae) was only little affected by residues of this pyrethroid, and thus could limit populations of the aphid Myzus persicae (Sulzer) (Homoptera: Aphididae) efficiently (Desneux et al. 2005).

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To summarize, a plant cuticle that offers resistance to a plant should comprise different properties. For efficient direct defense, it should have a surface microstructure on which herbivores are unable to walk, and eggs cannot attach; feeding or oviposition stimulants, and specifically nutrients, should be covered by cuticular waxes and not be present on the outer surface. For indirect defense, the microstructure should enable carnivores to hunt effectively; the sorption for attractants or stimulants for predators or parasitoids should be optimal; the visual appearance of the plant should betray the presence of feeding herbivores to the enemies. However, as the different species of herbivores and carnivores have various adaptations to specific cuticle characteristics, this optimal cuticle will probably never be feasible.

5.6 Perspectives In almost all plant-insect relationships, the cuticle as the first contact zone can certainly be expected to play a significant role for host acceptance or rejection by herbivores. However, for only few genera of plants have the plant-insect interactions that are mediated at the cuticular surface been studied well (e.g., Pisum, Brassica, Macaranga, Nepenthes, Eucalyptus). In these investigations, either glossy or waxy genotypes were compared, or manipulative experiments were carried out to assess the role of specific plant cuticle structures and chemistry. The different methods for investigating the role of the plant cuticle for insects have recently been reviewed (Müller 2006). Aspects of plant resistance mechanisms mediated by the plant cuticle are specifically important for plant breeding programs (Pelletier and Dutheil 2006). Also, for genetically modifying plants, cuticle characteristics and their barrier function need to be carefully evaluated (Kerstiens et al. 2006). However, this subject is quite complex, as the epicuticular waxes can have positive or negative effects on herbivorous and predatory insect species, or could decrease susceptibility to one pest but simultaneously increase susceptibility to another pest species. Many questions are still open, specifically with regard to induced plant responses at the plant surface: What happens morphologically at the plant cuticle after pure contact with insect legs, mouthparts, antennae or the ovipositor? What changes chemically? Does signaling occur at/through the cuticle? Do induced changes of the cuticle finally affect herbivorous insects, either the individual itself, conspecifics, or individuals of another species? It is surprising that there is so little knowledge on the very first contact zone of the plant for an approaching insect – a goldmine for future studies!

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Traw MB, Dawson TE (2002) Differential induction of trichomes by three herbivores of black mustard. Oecologia 131:526–532 Uematsu H, Sakanoshita A (1989) Possible role of cabbage leaf wax bloom in suppressing Diamondback moth Plutella xylostella (Lepidoptera: Yponomeutidae) oviposition. Appl Entomol Zool 24:253–257 Valkama E, Koricheva J, Ossipov V, Ossipova S, Haukioja E, Pihlaja K (2005) Delayed induced responses of birch glandular trichomes and leaf surface lipophilic compounds to mechanical defoliation and simulated winter browsing. Oecologia 146:385–393 van Dam NM, Hadwich K, Baldwin IT (2001) Induced responses in Nicotiana attenuata affect behavior and growth of the specialist herbivore Manduca sexta. Oecologia 122:371–379 van Loon JJA, Blaakmeer A, Griepink FC, van Beek TA, Schoonhoven LM (1992) Leaf surface compound from Brassica oleracea (Cruciferae) induces oviposition by Pieris brassicae (Lepidoptera, Pieridae). Chemoecology 3:39–44 Vogelmann TC (1993) Plant-tissue optics. Annu Rev Plant Physiol Plant Molec Biol 44:231–251 Vötsch W, Nicholson G, Müller R, Stierhof YD, Gorb S, Schwarz U (2002) Chemical composition of the attachment pad secretion of the locust Locusta migratoria. Insect Biochem Molec Biol 32:1605–1613 Vrieling K, Derridj S (2003) Pyrrolizidine alkaloids in and on the leaf surface of Senecio jacobaea L. Phytochemistry 64:1223–1228 Walton TJ (1990) Waxes, cutin and suberin. In: Harwood JL, Boyer J (eds) Lipids, membranes and aspects of photobiology. Academic Press, London, pp 105–158 Welke B, Ettlinger K, Riederer M (1998) Sorption of volatile organic chemicals in plant surfaces. Environ Sci Technol 32:1099–1104 Whitehouse P, Holloway PJ, Caseley JC (1982) The epicuticular wax of wild oats in relation to foliar entry of the herbicides diclofop-methyl and difenzoquat. In: Cutler DF, Alvin KL, Price CE (eds) The plant cuticle. Academic Press, London, pp 315–330 Wollenweber E (1989) Exudate flavonoids in flowering plants and ferns. Naturwissenschaften 76:458–463 Wollenweber E, Christ M, Dunstan RH, Roitman JN, Stevens JF (2005a) Exudate flavonoids in some Gnaphalieae and Inuleae (Asteraceae). Z Naturforsch C Biosci 60:671–678 Wollenweber E, Dorr M, Bohm BA, Roitman JN (2004) Exudate flavonoids of eight species of Ceanothus (Rhamnaceae). Z Naturforsch C Biosci 59:459–462 Wollenweber E, Dorsam M, Dorr M, Roitman JN, Valant-Vetschera KM (2005b) Chemodiversity of surface flavonoids in Solanaceae. Z Naturforsch C Biosci 60:661–670 Woodhead S (1983) Surface chemistry of Sorghum bicolor and its importance in feeding by Locusta migratoria. Physiol Entomol 8:345–352 Woolley JT (1971) Reflectance and transmittance of light by leaves. Plant Physiol 47:656–662 Yang G, Isenhour DJ, Espelie KE (1991) Activity of maize leaf cuticular lipids in resistance to leaf-feeding by the fall armyworm. Florida Entomol 74:229–236 Yencho GC, Renwick JAA, Steffens JC, Tingey WM (1994) Leaf surface extracts of Solanum berthaultii Hawkes deter Colorado beetle feeding. J Chem Ecol 20:991–1007 Zabkiewicz JA (2000) Adjuvants and herbicidal efficacy – present status and future prospects. Weed Res 40:139–149 Zalucki MP, Clarke AR, Malcolm SB (2002) Ecology and behavior of first instar larval Lepidoptera. Annu Rev Entomol 47:361–393

Chapter 6

Wound-Periderm Formation Idit Ginzberg

Herbivores, and particularly chewing insects, cause substantial damage to the plant. In addition to lost tissue, there are great concerns of pathogen invasion and water loss at the site of the attack. One of the plant’s defense strategies is the formation of wound periderm at the boundaries of the invaded or damaged region to isolate it from non-wounded healthy tissue. The development of wound periderm following insect feeding has never been specifically examined; although studies of herbivory and wound signaling have indicated extensive overlap in the respective sets of induced genes. The periderm protective characteristics are mainly due to the suberized walls of its outer cell layers. Suberin is composed of aromatic and aliphatic polyester domains, and associated waxy material, providing biochemical and structural barriers against pathogen infection, and contributes to water-proofing of the periderm. Most of the current knowledge on wound periderm derives from healing processes of mechanically wounded potato tubers. The review summaries these studies, in light of plant response to herbivory.

6.1 Introduction Aerial plant organs are generally protected by the cuticle and waxes that separate the outer environment and the interior tissues, and help maintain internal homeostasis. Upon injury of plant surfaces, the inner tissues are exposed to the surroundings, and may become desiccated and/or infected by pathogens, unless impermeability is rapidly reestablished. Plants do not heal wounds in the sense that damaged tissue is completely restored; instead, wound healing involves minimizing pathogen invasion and fluid loss. Although wound responses may differ among plant species and organs, they follow a similar pattern. The first layer of intact cells adjacent to the wound is modified by the accumulation of various antimicrobial and water-impermeable I. Ginzberg Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel e-mail: [email protected] A. Schaller (ed.), Induced Plant Resistance to Herbivory, C Springer Science+Business Media B.V. 2008

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substances, including lignin and suberin (Rittinger et al. 1987), to form a sealing layer. This may be followed by the development of periderm tissue under the sealing layer (Lulai and Freeman 2001; Rittinger et al. 1987; Thomson et al. 1995). Periderm is a protective tissue of secondary origin that replaces the epidermis when the latter is damaged. The protective characteristics of the periderm are mainly due to the deposition of suberin macromolecules on the walls of its outer cell layers. Periderm formation is a common phenomenon in stems and roots of dicotyledons and gymnosperms which increase in thickness by secondary growth, as well as in lenticels and following wounding. Actually, periderm formation and suberization processes are considered one of the generalized responses to wounding, irrespective of the nature of the natural protective polymer of the plant tissue (Bloch 1941; Dean and Kolattukudy 1976; Hawkins and Boudet 1996; Kolattukudy 2001; Rittinger et al. 1987). Almost all herbivores, and particularly chewing insects, cause substantial injury at the site of attack. In addition to the immediate and local damage to the plant due to lost tissue, there is the great concern of water loss from the perimeter of the injury (Aldea et al. 2005). One of the plant’s defense strategies is the formation of wound periderm at the boundaries of the invaded or damaged region to isolate it from non-wounded healthy tissue (Franceschi et al. 2005). The wound periderm may prevent successive pest invasions (Ichihara et al. 2000) and wall off pathogen infection. However, the development of wound periderm following insect feeding has never been specifically studied. Studies of herbivory and wound signaling have indicated extensive overlap in the respective sets of induced genes (Major and Constabel 2006; Ralph et al. 2006; Reymond et al. 2000), and insect feeding has been simulated using mechanically wounded plants, but such experiments are typically too short for periderm to form. Most of our current knowledge on wounding periderm and suberization is derived from wound-induced healing processes in potato tuber. Wound-periderm formation is induced by removing the tuber skin or excising discs of tuber flesh using a cork borer, and allowing the exposed tissue to heal. This procedure enables the isolation of significant amounts of suberized periderm which are then available for anatomical and chemical characterization, as well as for direct measurements of water permeability. In addition to the technical advantages involved in using the potato-tuber-disc system, there is a resemblance between the wound periderm of potato and that of other plants. Tissue development and anatomy are highly similar, and chemical components of suberin from other species are also found in the suberized periderm of potato (Gandini et al. 2006; Schreiber et al. 2005). Collectively, these characteristics of induced wound periderm in potato tubers make them a good model to study suberized wound-periderm tissue in plants.

6.2 Potato Periderm: Origin and Anatomy Periderm tissue replaces the epidermis early in potato-tuber development (Reeve et al. 1969). This type of periderm, designated natural or native periderm, is similar to wound periderm in terms of tissue origin, structure and morphology (Sabba and

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Lulai 2002). Both periderm types consist of similar chemical components, although they differ in their relative quantities (Lapierre et al. 1996; Negrel et al. 1996). The native periderm is initiated by divisions of both the epidermal and subepidermal cells, first at the stem end and later in the bud-end region of the tuber (Artschwager 1924; Reeve et al. 1969). New periderm is continuously added by cell divisions during tuber maturation. The capacity to produce new periderm upon wounding is retained by all parenchyma tissues in the tuber, but it is most pronounced close to the original, native periderm (Artschwager 1927). Both native and wound periderms consist of three cell types: the external suberized phellem cells derive from a single-cell meristematic layer, the phellogen (cork cambium), localized underneath them; inward cell divisions of the phellogen give rise to the parenchyma-like phelloderm (Fig. 6.1). Following suberization of their walls, the phellem cells die, thus creating the outer protective layer. The term periderm and those referring to its components are derived from Greek words, among which phellem means cork, gen, to produce, derma, skin, and peri, about (Esau 1965). In non-wounded tubers, the suberized phellem constitutes the potato skin (Lulai and Freeman 2001). In both wounded and native periderm, the phellem cells are identified by their rectangular shape and arrangement in columnar rows and by the fact that their walls autofluoresce under UV illumination (Fig. 6.1). This columnar pattern is a result of their origin from periclinal divisions in the phellogen cell layer (Artschwager 1924); the autofluorescence is due to the presence of aromatic suberin polymers in their cell walls (Bernards and Lewis 1998). In immature periderm (both native and wounding), the actively dividing phellogen is labile and prone to fracture, allowing separation of the suberized phellem from the underlying phelloderm and parenchyma cells (Lulai and Freeman 2001; Sabba and Lulai 2002). When the phellogen’s meristematic activity ceases, the cell walls thicken and become more resistant to fracture. Unesterified pectins may be involved in cell–cell adhesion via calcium-pectate formation (Kobayashi et al. 1999; Parker et al. 2001), and they have been detected in maturing native periderm (Lulai and Freeman 2001; Sabba and Lulai 2002, 2005). However, maturation of wound periderm is not associated with an increase in unesterified pectin in the phellogen walls (Sabba and Lulai 2002, 2004). Nevertheless, to gain maximal protection in the wounded area, a mature wound periderm should develop in which phellogen activity ceases and the suberized phellem layers adhere strongly to the underlying phelloderm.

6.3 Chemical Composition of the Periderm 6.3.1 Suberin 6.3.1.1 Occurrence The periderm owes its protective characteristics to the suberized walls of the phellem cells. Identifying suberized walls in a variety of plant organs has led to

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Native periderm

Phellem (skin) Phelloderm

B

Wound periderm Wounding by skin removal

1–2d

3d

4–5d

6d

Fig. 6.1 Potato native and wound periderm. Cross sections of potato (Solanum tuberosum L. cv. Desiree) tuber surface were stained with Safranin O/Fast Green and viewed with light (left panel) and UV (right panel) microscopes to examine tissue morphology and autofluorescence of suberized cells, respectively. (A) Native periderm of mature tuber with suberized phellem cells (the ‘skin’) and the parenchyma-like phelloderm, (B) Development of wound periderm following removal of the skin. Wounding induced suberization of the exposed tuber cells (1–3d). On the third day a meristematic phellogen that appears as multinuclear region developed below these cells (circled). Outward divisions of the phellogen produced the characteristic suberized phellem layers (4–6d). The dark grains are amyloplasts. Bar represents 100 ␮m


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the conclusion that suberization of cell walls is a universal mechanism that plants use whenever there is a need for the cell wall to become an effective diffusion barrier (Dean and Kolattukudy 1976; Kolattukudy 1984, 2001). Examples include epidermis, hypodermis and endodermis in roots (Schreiber et al. 1999; Zeier et al. 1999; Zimmermann et al. 2000) and in other underground plant organs, such as tubers and stolons (Espelie et al. 1980), as well as suberization of chalazal cells (Cochrane et al. 2000), the bundle sheath (Evert et al. 1996), and the base of secretory glands (Bakker and Baas 1993), where compartmentalization of nutrients or toxic materials is required. One specific example is the suberized walls of phenol-storing cells which are part of the plant’s constitutive defense mechanism against grazing animals and feeding insects. The toxic compounds are kept compartmentalized in a reduced, poised state within the cell vacuoles, while at the same time providing the means for their rapid oxidation, should decompartmentalization occur, e.g. via wounding (Beckman 2000). These phenolic compounds can be further used as building blocks for the suberin macromolecules as one means of sealing off the immediate site of cellular penetration. If the stress persists, these same processes promote the prolonged build-up of indole acetic acid (IAA) and ethylene, resulting in a cascade of events that produce a peridermal defense at depth, several centimeters below the point of immediate danger (Beckman 2000). In addition to its developmentally regulated biosynthesis, suberin synthesis is induced and its polymers are deposited in the walls of cells adjacent to wound sites during wound healing. Most importantly, suberization develops not only on tubers and other organs that are normally protected by suberin but also on fruits and leaves, which are normally protected by cutin (Bakker 1988; Dean and Kolattukudy 1976; Keren-Keiserman et al. 2004; Simons and Chu 1978). 6.3.1.2 Cellular Localization Suberin is composed of aromatic and aliphatic polyester domains, as well as associated waxy material, and is localized between the primary wall and the plasmalemma (Kolattukudy 1984). The model for suberin macromolecules suggests that the aromatic domains are attached to the cell-wall carbohydrates (Yan and Stark 2000), and the aliphatic domains are attached covalently to the phenolic domain via ester bonds (Bernards and Lewis 1992; Kolattukudy 1980, 1984). Transmission electron microscopy of suberized walls shows a lamellar structure in which the light segments probably constitute the aliphatic domain (Soliday et al. 1979). The suberin in potato wound periderm is deposited from the oldest, outer cell layer inward. Deposition occurs in a segmented fashion within each cell layer, first on the outer tangential cell walls, then on the associated radial walls and finally, on the inner tangential walls (Lulai and Corsini 1998). Suberin structure and chemical composition, as well as the enzymes involved in its biosynthesis, have been reviewed in detail (Bernards 2002; Bernards and Lewis 1998; Gandini et al. 2006; Graça and Santos 2007; Kolattukudy 2001). In the present review, this information is given in brief, to demonstrate the complexity of the suberization process and the difficulties in deciphering its structure and composition, which to date have only been partially characterized.

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6.3.1.3 Purification and Detection As suberin is deposited within the cell wall and attaches to it, it is impossible to isolate pure suberin from the cell wall. In the case of healing potato discs, the periderm tissue is peeled away from the tuber flesh and the adhering carbohydrates are removed by treatment with a mixture of hydrolases, such as cellulase and pectinase (Kolattukudy and Agrawal 1974; Vogt et al. 1983), leaving behind an insoluble material which is enriched in suberized walls. Further hydrolysis of noncellulosic polysaccharides is possible using trifluoroacetic acid (Arrieta-Baez and Stark 2006). The soluble waxes associated with the polymer are then extracted with organic solvents and analyzed by gas chromatography/mass spectrometry (GC/MS). Aliphatic components are released from the polymer by depolymerization with alkaline hydrolysis, transesterification with methanol in the presence of BF3 or NaOCH3 , or exhaustive hydrogenolysis with LiAlH4 in tetrahydrofuran (Kolattukudy 1984). The ether and C–C bonds in the aromatic polymer are oxidatively cleaved with alkaline nitrobenzene, CuO or thioglycolate treatment (Kolattukudy 1984). The derivatized suberin monomers are identified by GC/MS, high-pressure liquid chromatography (HPLC) or nuclear magnetic resonance (NMR; Kolattukudy 1984; Yan and Stark 2000).

6.3.1.4 Chemical Composition and Biosynthesis The aromatic domain of potato-periderm suberin is composed of hydroxycinnamates and their amide derivatives as well as monolignols (Bernards and Lewis 1998; Yan and Stark 2000). The major components of the aliphatic domain are ␣, ␻-diacids and ␻-hydroxyacids, with minor amounts of alkan-1-ols and alkanoic acids (Graça and Pereira 2000). Glycerol has been suggested to be involved in the cross-linking between the corresponding polyesters, allowing the formation of a three dimensional network (Graça and Pereira 2000), and recently it was shown that glycerol-3-phosphate acetyltransferase5 gene play a critical role in suberized cell wall biogenesis (Beisson et al. 2007). In addition, the potato periderm contains up to 20% extractable lipids (waxes). Besides linear long-chain aliphatic wax compounds, alkyl ferulates have been detected as significant constituents; in wound periderm they amount to more than 60% of the total extracts (Schreiber et al. 2005). It is worth noting that suberin aromatic and aliphatic domains differ from lignin and cutin, respectively. The major distinction between cutin and suberin is that the former contains polar in-chain hydroxylated and/or epoxy acids as major components (Matzke and Riederer 1991). As for lignin, it is comprised of polymerized hydroxycinnamyl alcohols, i.e. monolignols (Bernards and Razem 2001; Bernards et al. 2000). The phenylpropanoid pathway is induced in both aromatic suberin and lignin biosynthesis. The activities of phenylalanine ammonia-lyase (PAL), 4-coumaryl-CoA ligase (4-CL) and cinnamyl alcohol dehydrogenase (CAD) increase in both suberization and lignification, while cinnamoyl-CoA oxidoreductase (CCR) activity is induced only upon initiation of lignification (Bernards et al. 2000). The lack of CCR induction in suberizing potato periderm indicates channeling of hydroxycinnamoyl-CoA


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derivatives away from monolignol formation and toward that of suberin derivatives (Bernards et al. 2000). Hydroxylation and methylation of hydroxycinnamic acids occur at the hydroxycinnamoyl-CoA derivatives level, and putatively suberin-specific O-methyltransferase has been cloned from maize (Held et al. 1993). Channeling of the phenylpropanoid phenolics into suberin has been suggested to involve the activity of tyramine hydroxycinnamoyl transferase (THT; Negrel et al. 1993, 1996), and its cDNA has been cloned from potato (Schmidt et al. 1999). It has further been suggested that the phenolic domain is polymerized via peroxidase activity (Cottle and Kolattukudy 1982) and requires H2O2 (Razem and Bernards 2003), and a suberizationassociated anionic peroxidase has been cloned (Roberts et al. 1988; Roberts and Kolattukudy 1989). Cationic isoforms may also be involved, although these have not yet been cloned (Quiroga et al. 2000). Reactions which are specific to aliphatic suberin include fatty-acid elongation (C-18 and C-24 to C-32) and oxidation. Ketoacyl-CoA synthase, catalyzing the first step in the elongation of 18:0 via malonyl-CoA, has been cloned from suberizing primary roots of maize (Schreiber et al. 2000). The activities of two NADP-dependent dehydrogenases that catalyze the oxidation of ␻-hydroxyacids and ␻-oxoacid to their corresponding dicarboxylic acids are considered to be key steps in aliphatic suberin biosynthesis (Agrawal and Kolattukudy 1977); however, their corresponding cDNAs have not yet been cloned. The incorporation of ferulates into suberin polymers has been suggested to be through conjugation to the hydroxyfatty acids by the hydroxyfatty acid feruloyl-CoA transferase (HHT; Lofty et al. 1994). Extensive chemical analysis of suberin has enabled the identification of its monomeric and oligomeric fragments and the construction of a conceptual model of its structure in the cell wall. Nevertheless, only sketchy details, described herein, are known regarding the biosynthesis of suberin-specific components, the polymerization reactions, the bonding between the aromatic and aliphatic domains and the anchoring of the macromolecules to carbohydrates in the primary cell wall. This and the regulation of suberin induction and biosynthesis await further clarification.

6.4 Protective Characteristics of Wound Periderm Following suberization of their cell walls, the phellem cells die, creating a protective layer that covers the previously exposed tissue at the wounding site. The corpses of the cells are filled with air and therefore provide thermal insulation, the suberized walls prevent invasion by microorganisms (mechanically and chemically), and wax deposits that are embedded within the suberin material prevent desiccation of the previously exposed plant tissue (Kolattukudy 1977). Mechanisms by which suberin may impart disease resistance have been suggested by Kolattukudy (1984): it may act as a barrier to the diffusion of pathogen enzymes or toxins into living tissues, as a structural barrier to pathogen ingression, or as a biochemical barrier to microbes due to the high proportion of phenolic materials incorporated into the suberin polymer. In potato tubers, synthesis and integration into the cell walls of hydroxycinnamic acid amides of tyramine and octopamine constitute a very early response

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to wounding (Negrel et al. 1993). These amides exhibit antifungal activity and are postulated to contribute to the formation of a primary phenolic barrier against pathogen invasion, and to be the building blocks, with other phenolics, of the suberin polyaromatic domain which then serves as a permanent barrier (Bernards et al. 1995), replacing the cuticle. The suberin aromatic domain further strengthens the cell walls against fungal enzymatic hydrolysis (Negrel et al. 1995), and the thickness of the suberized cell layers is highly correlated with resistance to pathogens (Morris et al. 1989). These postulated roles of the suberin phenolic domain were supported by experiments with inhibitors of suberin phenolic synthesis which prevented the development of pathogen resistance (Bostock and Stermer 1989; Hammerschmidt 1984). Lulai and Corsini (1998) further suggested that the suberin phenolic domain provides resistance to bacterial (Erwinia carotovora subsp. carotovora) but not fungal (Fusarium sambucinum) infection, whereas deposition of the suberin aliphatic domain is responsible for the latter. In potato-tuber wound-healing tissue, the deposition of suberin aliphatic components has also been correlated with the development of resistance at the tissue’s surface to diffusion of water vapor (Kolattukudy and Dean 1974). However the waxes that associated with the suberin polymer were suggested to play the crucial role in water-proofing of the periderm. Specific inhibition of wax synthesis inhibited the development of resistance to water vapor diffusion at the periderm surface, indicating that the waxes associated with the suberin polymer, rather than the polymer itself, constitute the major diffusion barrier formed during wound healing (Soliday et al. 1979). This role of suberin-associated waxes is analogous to the contribution of waxy material to the aerial cuticle: in cuticles, water permeability is completely determined by the waxes (Schönherr 1976). Extraction of the wax from the periderm results in an increase in water permeability (Espelie et al. 1980; Schreiber et al. 2005; Vogt et al. 1983). Interestingly, water permeability of the wound periderm is, on average, 100 times higher than that of natural periderm (Schreiber et al. 2005; Vogt et al. 1983). Schreiber et al. (2005) correlated the formation and amount of the aliphatic domain in potato-tuber periderm with its efficiency as a barrier to water transport. The differences in suberin chemical composition and amounts between native and wound periderm were minor compared to the large differences in water permeability, suggesting that the molecular arrangement and precise localization of the deposition of suberin and its associated waxes within the cell wall contribute to its efficiency as a barrier to water transport.

6.5 Erecting the Periderm Barrier 6.5.1 Time Course of Periderm Development Following wounding, the erection of a protective barrier before pathogen invasion and establishment is fundamental to host resistance. Hence, the rate of periderm


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development and suberization of its outer phellem cells are important determinants in plant recovery after wounding. Cytological events that may lead to phellogen initials and periderm development have been suggested to follow a transient increase in IAA and lipid hydroxyperoxide, both reaching maximal levels 20–30 min post-wounding of potato tubers (Fabbri et al. 2000). This is followed by mitotic activity of cells competent to produce periderm, starting from 120 min post-wounding (Fabbri et al. 2000). Cell divisions are accompanied by a biphasic increase in protein synthesis, 2 h and then between 12 and 24 h post-wounding (Morelli et al. 1994), and with an increase in polymerized actin and microfilament bundles in cells at the wounding site, 12 h post-wounding (Morelli et al. 1998). The depositions of suberin aliphatic and aromatic compounds in the developing periderm have been found to be separate processes (Lulai and Morgan 1992). Suberin polyphenolics accumulate first during wound-induced suberization, while the suberin aliphatics accumulate last and complete the suberin barrier (Lulai and Corsini 1998; Lulai and Morgan 1992). PAL activity, which is a necessary prerequisite for the biosynthesis of suberin aromatic monomers, was found to reach a transient peak 12 h after potato-tuber wounding; however, it was not restricted to the suberizing cell layers, and was also detected in deeper layers (Borchert 1978). Suberin deposition initiated 24 h after slicing the potato tuber (Thomas 1982). When radioactive cinnamic acid was fed to wound-healing potato-tuber discs, maximal uptake was measured during the first two days post-wounding and declined thereafter (Cottle and Kolattukudy 1982b). Consecutively, the highest deposition of the phenolic suberin components onto the cell wall was measured between three and seven days of wound healing (Cottle and Kolattukudy 1982b; Hammerschmidt 1985; Lulai and Corsini 1998). Accordingly, total peroxidase activity, presumed to be involved in polymerization of the aromatic domain, and its anchorage to the cell wall, increased gradually in the suberizing cells four days post-wounding and remained high thereafter (Borchert 1978). The suberin phenolic domain provides esterification sites for the aliphatic components; hence, initiation of aliphatics deposition in the periderm tissue was detected upon completion of suberin phenolic depositions at the first, outer layer of the phellem (Lulai and Corsini 1998). With the use of cycloheximide and actinomycin D, it was demonstrated that RNA and protein synthesis of ␻-hydroxyacid dehydrogenase, the enzyme involved in the formation of suberin aliphatic components, occurs between 72 and 96 h post-wounding (Agrawal and Kolattukudy 1977). Measurement of the accumulation of the aliphatic components indicated a slower increase up to five days post-wounding followed by a dramatic increase up to day ten of suberization (Agrawal and Kolattukudy 1977; Lulai and Corsini 1998). The temporal incorporation of [1−14C]oleic acid and [1−14C]acetate into aliphatic components of suberin supported this time course (Dean and Kolattukudy 1977). Thus, in potato, deposition of the suberin components initiates one to two days post-wounding, and formation of fully protective tissue is complete about ten days later.

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The above time course of suberin accumulation up to the formation of a fully protective barrier may vary with potato genotype (Lulai and Orr 1995). Such variation is also found in woody plants, e.g. suberin accumulation in peach bark wounds was found to be highly cultivar-dependent (Biggs and Miles 1988).

6.5.2 Physiological Factors that Determine the Rate of Wound Healing The above-described time course for the development of a suberized periderm barrier in the potato-tuber-disc system was determined under controlled conditions; however, physiological as well as environmental conditions influence the capacity to heal wounds. The effect of temperature and relative humidity on periderm formation has been extensively studied in the context of the industrial/agricultural practice of curing skinning injuries of newly harvested potato tubers. Histological observations indicated that wound-induced periderm formation occurs most rapidly at 25◦ C; wound healing was delayed at lower temperatures (10◦ C–15◦ C), while temperatures as high as 35◦ C prevented periderm formation (Morris et al. 1989; Thomas 1982). Similarly, in wound-healing peach bark, phellogen regeneration was strongly influenced by temperature and occurred more rapidly under moderate climatic conditions (Biggs and Miles 1988). Nevertheless, high humidity (98%) in combination with the optimal temperature (25◦ C) hastened wound-induced periderm formation in potato tubers (Morris et al. 1989). The effect of temperature on wound-induced suberin formation was determined in the potato-tuber-disc system by monitoring the development of water-loss resistance. The optimum temperature for the formation of aliphatic suberin monomers and the development of resistance to water-vapor conduction was 26.4◦C, whereas alkane synthesis was optimal at 18.6◦C (Dean 1989). Lower temperatures reduced suberin monomer production. The difference in optimal temperatures for the synthesis of suberin aliphatic and wax components may derive from the temperature sensitivity of fatty-acid-chain-elongation reactions: C21 alkane dominates the alkanes found at 19.5◦C whereas at 29.4◦C, the three homologs C21 , C23 and C25 , are found in equal proportions (Dean 1989). Temperature dependence of periderm development and suberin biosynthesis implies an effect of seasonal variation on the plant’s ability to recover from injury. Wound depth affects wound-healing rate: shallow tuber wounds begin to heal faster than deeper ones (Lulai and Orr 1995). The age of the wounded tissue also affects the healing process. Kumar and Knowles (2003) showed that aging potato tubers lose the capacity to heal wounds during storage, together with their ability to sustain superoxide production on the wound surface. They suggested that high activity of superoxide dismutase (SOD) in the aging tissues limits the accumulation of superoxide radicals, thereby reducing the wounded tissue’s ability to oxidize 1-aminocyclopropane-1-carboxylic acid (ACC) to wound-induced ethylene. The lower availability of superoxide radicals may further affect the rate of the protective


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layer’s evolution as they are required for polymerization of the suberin aromatic domain (Bernards and Razem 2001; Razem and Bernards 2003). In addition, older tubers exhibit a reduced ability to increase PAL activity in response to wounding, which potentially limits the availability of suberin aromatic precursors and further contributes to age-induced loss in wound-healing ability and pathogen resistance (Kumar and Knowles 2003).

6.5.3 Hormonal Involvement Suberization, as measured by resistance of the tissue surface to water-vapor diffusion, was inhibited by millimolar concentrations of IAA, severely inhibited by micromolar concentrations of cytokinin, but stimulated by abscisic acid (ABA; Lulai and Orr 1995; Soliday et al. 1978). The involvement of ABA in wound healing was first observed when the hormone was released into the washing medium within one day of cutting the potato discs; addition of ABA to the incubation medium of the washed tuber discs reversed the washing-induced inhibition of suberization (Soliday et al. 1978). It was therefore proposed that during the early phase of wound healing, ABA plays a role in triggering a chain of biochemical processes which eventually result in the formation of a suberization-inducing factor, responsible for the induction of enzymes involved in suberin biosynthesis. The effect of ABA on suberization was further studied in potato-tuber tissue culture by measuring the deposition of suberin components. ABA treatment resulted in an increase in both aromatic and aliphatic polymeric components of suberin, as well as hydrocarbons and fatty alcohols characteristic of waxes associated with potato suberin, and certain enzymes postulated to be involved in suberization (Cottle and Kolattukudy 1982). As ethylene is a well-known wound-response hormone (Abeles et al. 1992; O’Donnell et al. 1996), its role in wound-induced suberization of potato tuber was examined over a 9-day wound-healing period, using a variety of inhibitors of ethylene biosynthesis and action (Lulai and Suttle 2004). Ethylene evolution was stimulated by wounding and reached a maximum two to three days post-wounding, after which it gradually declined. However, the hormone was not required for woundinduced accumulation of suberin polyphenolics in the sealing layer (i.e., suberization of existing cells at the wound surface) during the first two to four days of wound healing, or for the subsequent suberization of phellem cells (between four and nine days; Lulai and Suttle 2004). Although the effects of ABA, IAA and ethylene were examined with respect to the suberization processes, their involvement in the evolution of wounding periderm, i.e. the initiation of phellogen initials, regulation of cell division, and determination of phellem and phelloderm fate, remained unclear. The time course of periderm formation and its suberization may differ in planta, and may depend on various physiological and environmental stimuli; nevertheless, the rate and extent of suberin accumulation are more important than the actual number of new phellem cells or the thickness of the new suberized layers. This means that heavily suberized periderm composed of relatively few cell layers may

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be a more effective barrier to pathogen ingression than a thicker periderm (i.e., one with more cell layers) that is relatively less suberized (Biggs 1989; Biggs and Miles 1988).

6.6 Perspectives Wound-periderm formation and suberization of cell walls at the wounding site are fundamental features of the plant’s response to disruption of its natural outer protective membranes. The rate of establishment of suberized periderm following injury is an important factor in the plant’s resistance to indirect damage following the wounding, such as water loss and pathogen invasion. However, the regulation, developmental stages and biosynthetic pathway of periderm development and suberization are still not completely understood. Moreover, herbivore feeding differs from plain injury as the former secrete saliva which may interact with chemical factors involved in the healing process. The effect of the plant-herbivore interaction and the resultant signaling with respect to suberized periderm development has not been considered experimentally, and requires further clarification.

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Soliday CL, Dean BB, Kolattukudy PE (1978) Suberization: inhibition by washing and stimulation by abscisic acid in potato disks and tissue culture. Plant Physiol 61:170–174 Soliday CL, Kolattukudy PE, Davis RW (1979) Chemical and ultrastructural evidence that waxes associated with the suberin polymer constitute the major diffusion barrier to water-vapor in potato tuber (Solanum tuberosum L). Planta 146:607–614 Thomas P (1982) Wound-induced suberization and periderm development in potato tubers as affected by temperature and gamma irradiation. Potato Res 25:155–164 Thomson N, Evert RF, Kelman A (1995) Wound healing in whole potato tubers: a cytochemical, fluorescence, and ultrastructural analysis of cut and bruise wounds. Can J Bot 73:1436–1450 Vogt E, Schonherr J, Schmidt HW (1983) Water permeability of periderm membranes isolated enzymatically from potato tubers (Solanum tuberosum L). Planta 158:294–301 Yan B, Stark RE (2000) Biosynthesis, molecular structure, and domain architecture of potato suberin: a 13 C NMR study using isotopically labeled precursors. J Agric Food Chem 48:3298–3304 Zeier J, Ruel K, Ryser U, Schreiber L (1999) Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots. Planta 209:1–12 Zimmermann HM, Hartmann K, Schreiber L, Steudle E (2000) Chemical composition of apoplastic transport barriers in relation to radial hydraulic conductivity of corn roots (Zea mays L.). Planta 210:302–311

Chapter 7

Traumatic Resin Ducts and Polyphenolic Parenchyma Cells in Conifers Paal Krokene, Nina Elisabeth Nagy and Trygve Krekling

Conifers integrate multiple constitutive and inducible defenses into a coordinated, multitiered defense strategy. Constitutive defenses, established before an attack, represent a fixed cost and function as an insurance against inevitable attacks. Inducible defenses, mobilized in response to an attack, represent a variable resistance that is turned on when it is needed. Polyphenolic parenchyma cells (PP cells) that are specialized for synthesis and storage of phenolic compounds are abundant in the phloem of all conifers. In addition to being a prominent constitutive defense component, PP cells are also involved in a range of inducible defense responses, including activation of existing PP cells, production of new PP cells, and wound periderm formation. Their abundance and varied defensive roles make the PP cells the single most important cell type in conifer defense. Another important defense are traumatic resin ducts which are induced in many conifers after various biotic or abiotic challenges. Traumatic resin ducts are primarily formed in the xylem where they appear in tangential rows, but inducible resin ducts are also formed in the phloem of some conifers. Activation of PP cells and formation of traumatic resin ducts take place through the octadecanoid pathway, involving jasmonate and ethylene signaling.

7.1 Introduction The conifers include about 630 species of woody gymnosperms that are widely distributed, from Arctic and alpine timber lines to tropical forests (Farjon 2001). Even though the conifers are extremely species-poor compared to the enormous diversity of angiosperms, extant conifers are in many ways remarkably successful plants. Many species are the dominant plants over huge areas, and are major forest trees with immense ecological and economical values. Conifers grow taller, stouter, and older than any other trees, with record holders such as coast redwood Sequoia sempervirens (115.6 m tall), Montezuma cypress Taxodium mucronatum P. Krokene ˚ Norway Norwegian Forest and Landscape Institute, N-1432 As, e-mail: [email protected] A. Schaller (ed.), Induced Plant Resistance to Herbivory, C Springer Science+Business Media B.V. 2008

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(11.4 m diameter) and Great Basin bristlecone pine Pinus longaeva (4844 years). Taxonomically, the conifers constitute the division Pinophyta, which includes a single class (Pinopsida) with a single extant order (Pinales or Coniferales). The most species-rich and geographically widespread of the seven to eight recognized families of the Pinales is the Pinaceae with about 225 species, including important Northern Hemisphere forest trees in the genera Pinus, Picea, Larix and Abies. Other important conifer families are the Cupressaceae (∼135 species), the Podocarpaceae (∼185 species) and the Araucariaceae (41 species), all of which include many tropical and Southern Hemisphere species (Farjon 2001). A key to the conifer’s success are their effective defensive strategies that have enabled them to fend of attackers over a phylogenetic history of more than 200 million years. Long-lived and apparent plants such as conifers are bound to be found by natural enemies during ecological time, and thus need a wide range of defenses that are effective against both specialist and generalist enemies (Feeny 1976). This is achieved by integrating multiple constitutive and inducible defenses into a coordinated, multitiered defense strategy (Franceschi et al. 2005). Constitutive defenses, such as cork bark and preformed resin and phenols, act as a first line of defense that inhibits or slows down an initial attack, and if this is not sufficient, inducible defenses are mobilized to kill or compartmentalize the attackers. The temporal and spatial integration of the constitutive and inducible defenses probably contribute to reducing the total costs of defense. Two types of inducible defenses can be distinguished in conifers based on their level of organizational complexity. At the simplest level are defense responses that are a result of changed metabolism of existing cells, such as lignification of cell walls, induction of resin production in preformed resin ducts, and activation of the hypersensitive response. At a more complex organizational level are induced defense responses that involve changes in cell division and differentiation, leading to formation of e.g. traumatic resin ducts and a wound periderm. The constitutive and inducible defenses in conifers may be both mechanical and chemical in nature. Mechanical defenses consist of structural elements that provide toughness or thickness to tissues, and thereby interfere with penetration and degradation by attackers. Prominent mechanical defenses in conifer stems include the outer defense barrier (the periderm and cork bark), tough elements of the phloem (e.g., sclerenchyma and calcium oxalate crystals), and tissue impregnation with polymers (e.g., lignins and suberin) (Wainhouse et al. 1990; Kartuch et al. 1991; Franceschi et al. 2000, 2005; Hudgins et al. 2003b). Chemical defenses include substances with toxic or inhibitory effects, such as allelochemicals, proteins, and enzymes. Chemical defenses in conifers often consist of pools of stored chemicals (e.g., phenolics or terpenoids) that are released after attack (Phillips and Croteau 1999; Schmidt et al. 2005). There is an obvious overlap between different ways of classifying conifer defenses, as the same defense traits can have both chemical and mechanical properties, and be both constitutively expressed and inducible. Resin for example, a major defense component in many conifers, is produced constitutively and stored in preformed resin ducts, but its production can also be induced in preformed resin ducts (Ruel et al. 1998; Lombardero et al. 2000; Hudgins and

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Franceschi 2004), or in inducible traumatic resin ducts (Bannan 1936, Hudgins et al. 2003a, 2004). Furthermore, resin is chemically toxic to insects and fungi, as well as having mechanical defense properties by being sticky and exuded under pressure when attacking organisms are penetrating the bark (e.g., Raffa et al. 1985; Cook and Hain 1988). In this chapter we describe two important anatomically and chemically based defense components of conifer bark and sapwood. Parenchyma cells with characteristic polyphenolic content occur in the bark of all conifers and are the most abundant living cell type of the secondary phloem. These polyphenolic parenchyma cells (PP cells) are specialized for synthesis and storage of phenolic compounds, and due to their ubiquitous occurrence and varied defensive roles, they may be regarded as the single most important cell type in conifer defense (Franceschi et al. 2005). PP cells are produced during normal phloem development, but undergo extensive changes in response to attack, and are thus a major site of both constitutive and induced defenses (Franceschi et al. 1998, 2000). Another major defense component in conifers are various resin containing structures that may be constitutively present or induced in the bark and sapwood of many species. We will focus on traumatic resin ducts (TRDs) which, as their name implies, are induced after various biotic or abiotic challenges. TRDs in the strict sense are found in the xylem, where they appear as tangential rows, but inducible resin ducts are also formed in the phloem of some conifers (Hudgins et al. 2004). Induction of PP cells and TRDs has been associated with the phenomenon of systemic induced resistance (Bonello and Blodgett 2003) that has been described in only a few members of the Pinaceae so far (Reglinsky et al. 1989; Christiansen et al. 1999b; Krokene et al. 1999, 2000, 2003; Bonello et al. 2001, Bonello and Blodgett 2003), but that is likely to be widespread in conifers.

7.2 Polyphenolic Parenchyma Cells 7.2.1 Anatomy and Development of PP Cells Polyphenolic parenchyma cells seem to play a prominent role in the defense of all conifer families that have been studied (Franceschi et al. 1998, 2000; Hudgins et al. 2003a, 2004) (Fig. 7.1). Considering their abundance and importance in conifer bark, surprisingly little was published on their structure and function until 1998, when Franceschi and co-workers published the first in a series of papers on PP cell anatomy, development, and roles in constitutive and inducible defenses (Franceschi et al. 1998, 2000, 2005; Krekling et al. 2000, 2004; Krokene et al. 2003; Hudgins et al. 2003a, 2004; Nagy et al. 2004). Norway spruce has served as a model species for most of this research, but studies on other conifers in the Cupressaceae, Podocarpaceae, Araucariaceae and Taxaceae have demonstrated the general importance of PP cells in conifer defense (Hudgins et al. 2003a, 2004).

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Fig. 7.1 Three-dimensional line drawing showing the basic bark and sapwood anatomy of conifer stems, using Norway spruce as an example. Characterizing features are the multiple concentric layers of defensive structures in the bark, including the periderm and multiple layers of polyphenolic parenchyma cells, and the sapwood with axial and radial resin ducts

Microscopy examination of conifer bark reveals that conifer defenses are laid out in a concentric, multilayered design around the vascular cambium (Franceschi et al. 2005). This is exemplified in the Pinaceae where the PP cells are organized in characteristic concentric rows (one cell wide) in the secondary phloem, separated by 9–12 rows of sieve cells, and associated albuminous cells interspersed with a few scattered brachysclereids or stone cells (Franceschi et al. 1998; Krekling et al. 2000; Figs. 7.1 and 7.2A). In temperate conifers, the PP cell cylinders form annual rings in the phloem, since a new row is laid down at the beginning of every growth season (Alfieri and Evert 1973; Krekling et al. 2000; Fig. 7.3). Running perpendicular to the tangentially oriented PP cell layers, the radial rays span from the secondary phloem into the xylem. Close to the cambium they are usually uniseriate, consisting of ray parenchyma only, but they frequently become multiseriate in the older parts of the phloem with a central radial resin duct (Krekling et al. 2000; Fig. 7.3A). In the non-Pinaceae, PP cells and rays are organized similarly, but the intervening blocks of sieve cells are only a few cell rows thick (Fig. 7.4B), and normally intersected by an almost complete tangential row of sclerenchymatic fiber cells (Fig. 7.2B). The fiber cells have lignified secondary wall thickenings and serve as structural elements as well as mechanical defense (Hudgins et al. 2003a, 2004). The highly ordered arrangement of densely spaced fiber rows and PP cells probably


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Fig. 7.2 Cross-sections showing the concentric organization of tissues in the bark (cortex and secondary phloem) and in the xylem of two different conifers. (A) A young stem of balsam fir (Abies balsamea), representing the Pinaceae-type phloem anatomy, with two annual layers of polyphenolic parenchyma (PP) cells, separated by a thicker layer of sieve cells. Large axial resin ducts are found in the cortex. (B) Young stem of bald cypress (Taxodium distichum) showing typical non-Pinaceae phloem anatomy, with alternating rows of fiber cells and PP cells, separated by thin layers of sieve cells. Both PP cells and fiber cells are laid down in annual layers, with one row of cells per year. Bars, 100 ␮m

constitutes a formidable physical barrier to organisms that attempt to penetrate the phloem (Hudgins et al. 2004). The fiber cells develop from precursor cells that are distinct from PP cells and normally require two to three years to become fully lignified (Franceschi et al. 2005). The importance of PP cells in conifer defense is indicated by the facts that they are produced early in both primary and secondary growth, and that considerable resources are spent on their development and maintenance. PP cells are produced by the interfascicular cambium during the earliest stages of primary growth in Norway spruce, and by the second year of growth, regular PP cell layers are produced (Krekling et al. 2000; Fig. 7.2A). In seasonal climates, a new annual layer of PP cells forms soon after the cambium resumes activity after winter dormancy at the beginning of each growth season, from an undifferentiated cell layer that was produced towards the end of the previous growth season (Krekling et al. 2000). Interestingly, a new PP cell layer is also produced after other major disruptions of cambial activity in Norway spruce, such as insect or pathogen attack (Krekling et al. 2000, 2004; Krokene et al. 2003). Maturation of PP cells is a slow process that may take five years or more in Norway spruce (Krekling et al. 2000). During this process the cells increase in size and gradually become more rounded, the cell walls thicken, and the phenolic bodies become more extensive. Additional PP cells may develop from undifferentiated axial parenchyma cells in between the regular annual layers during the first five to eight years after formation (Krekling et al. 2000). As a consequence of the gradual increase in PP cell size during maturation, the surrounding sieve cells are progressively crushed and compacted and become non-functional (Figs. 7.3A and C). The rows of PP cells, however, stay alive for many years; in 100-year-old

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Fig. 7.3 Cross-sections of the secondary phloem of Norway spruce (Picea abies), showing the anatomy and organization of polyphenolic parenchyma (PP) cells. (A) Cross section from control tree showing the normal, constitutive tissue with concentric layers of PP cells separated by sieve cells. A large radial resin duct inside a multiseriate ray is traversing the phloem and the xylem. Clusters of heavily lignified stone cells are randomly distributed in the older parts of the phloem. These sclerenchyma cells are derived from PP cells as the phloem ages. Bar, 100 ␮m. (B) Cross section showing induction of wound periderm formation and PP cells in the bark after inoculation with the blue-stain fungus Ceratocysis polonica. The wound periderm consists of suberized and lignified cells. Below the wound periderm close to the cambium the PP cells are enlarged due to


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Norway spruce trees PP cell rows formed more than 70 year ago contained living cells (Krekling et al. 2000). PP cells in older phloem layers may undergo cell division, and this enables the tree to maintain intact layers of PP cells as the stem diameter increases during growth (Krekling et al. 2000). In addition, new PP cells are recruited in the older layers of the secondary phloem by differentiation of existing residual parenchyma cells or ray cell derivatives (Fahn 1990; Krekling et al. 2000). The phenolic material characterizing the PP cells is stored inside a large vacuole that may fill most of the cell volume (the phenolic body). The phenolic nature of the PP cells is inferred from their bright yellow fluorescence when exposed to blue light (450–490 nm; Fig. 7.4A), strong staining (and quenching of fluorescence) by osmium tetroxide and the periodic acid-Schiff procedure (Fig. 7.3), and the presence of phenylalanine ammonia lyase (PAL), a key enzyme in phenolic synthesis (Parham and Kaustinen 1976; Franceschi et al. 1998). The vacuolar content of the PP cells is highly dynamic and changes in appearance over the year, as well as after pathogen attack or mechanical wounding (Fig. 7.3D). In Norway spruce the density of the phenolic body increases during autumn and appears dense-staining and granular by mid-winter. During spring and summer the density decreases again and reverts to a homogenous, light-staining appearance by mid-summer (Krekling et al. 2000). Attempts have been made to classify PP cells into four types based on the appearance of the vacuolar content (Franceschi et al. 1998), but any relationship between vacuolar morphology and resistance remains unclear. In addition to being a major storage site for phenolic compounds, PP cells also accumulate and store carbohydrates, in the form of starch grains, and lipids (Murmanis and Evert 1967; Alfari and Evert 1973; Krekling et al. 2000; Fig. 7.4C). Starch reserves build up through the summer months in Norway spruce, are completely absent in the winter, and begin to accumulate again in the spring (Krekling et al. 2000). In addition to serving as energy reserves, the starch and lipid of the PP cells can also be used for rapid synthesis of defensive chemicals in response to attack. Since PP cells and ray cells make up the bulk of the living cells in the phloem they are probably involved in radial and circumferential transmission of signals that activate defense responses (Franceschi et al. 2000; Krekling et al. 2004). Consistent with this notion, there are numerous symplasmic cell connections (plasmodesmata) facilitating transport of signal molecules and nutrients between PP cells, between ray cells and adjacent PP cells, and between ray cells (Krekling et al. 2000). PP cell are also involved in production of important mechanical defenses that are found in the secondary phloem of all conifers (Franceschi et al. 2005).

Fig. 7.3 (continued) mobilization of their polyphenolic vacuolar content, whereas PP cells in the immediate vicinity of the wound periderm have emptied their content. Traumatic resin duct formation has been induced in the xylem. Bar, 100 ␮m. (C) Larger magnification of normal, nonactivated annual layers of PP cells in the bark. Four annual rows of PP cells are shown, separated by 10–12 layers of sieve cells. Bar, 50 ␮m. (D) Larger magnification of bark three weeks after inoculation with Ceratocystis polonica, showing PP cells that have mobilized their phenolic content and increased in size. The older layers of sieve cells are compressed into dense areas of cell walls by the swelling PP cells. Bar, 50 ␮m

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Fig. 7.4 Large magnification cross-sections of polyphenolic parenchyma (PP) cells. The PP cells are named after their phenolic vacuolar inclusions that cover most of the cell volume. (A) The polyphenolic material is emitting a characteristic bright, yellowish fluorescence when illuminated with blue light (450–490 nm). (B) PP cells in Pacific yew (Taxus brevifolia) viewed in the SEM microscope in a back-scattered mode, demonstrating how the cell walls are encrusted with extra-cellular calcium-oxalate crystals. Small extra-cellular crystals are characteristic of non-Pinaceae conifers, in contrast to the Pinacea species that have larger, intra-cellular crystals. (C) The PP cells store starch grains in their cytoplasm, clearly visible after staining with the carbohydrate specific Periodic acids Schiff’s procedure. Bars, 25 ␮m

The distribution of stone cells (sclereids) in the secondary phloem of Norway spruce suggests that these cells are derived from PP cells (Franceschi et al. 2005). Stone cells are massive, irregularly shaped cells with thick lignified cell walls that occur as single cells or in small clusters in all Pinaceae plus Araucaria (Franceschi et al. 2005). Due to their physical toughness, stone cells can be a deterrent to bark boring organisms (Wainhouse et al. 1990, 1997; Hudgins et al. 2004). In the Pinaceae modified PP cells are producing calcium oxalate crystals that are likely to interfere with bark penetration as well (Hudgins et al. 2003b; Franceschi et al. 2005; Fig. 7.4B). Unlike normal PP cells, these modified cells are dead at maturity and


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have suberized cell walls. Crystals of calcium oxalate may also occur in the vacuoles of some of the PP cells along with the phenolic material (Franceschi et al. 1998).

7.2.2 Inducible Responses in PP Cells In addition to being a prominent constitutive component in the secondary phloem, PP cells are also involved in a range of inducible defense responses, including activation of existing cells, production of extra PP cells, and wound periderm formation. Activation or swelling of existing PP cells begins a few days after an attack and results in a four-fold increase in the PP cell volume and a change in the appearance of the phenolic content (Franceschi et al. 1998, 2000, 2005; Krokene et al. 2003; Fig. 7.3). The dramatic increase in PP cell size leads to extensive compression of the surrounding sieve cells, and at least in the Pinaceae, which produce many layers of sieve cells per year, the induced phloem is transformed into dense blocks of cell walls separated by layers of swollen PP cells (Figs. 7.3B and D). In addition to serving as a physical barrier to penetration by invading organisms this combined cell wall/PP cell barrier appears to be reinforced by phenolics released from the induced PP cells and deposited in the surrounding sieve cell walls (Franceschi et al. 2000). New or extra PP cells may be induced to develop from undifferentiated parenchyma cells in between the regular annual PP cell layers. This is not part of the normal developmental program of the secondary phloem and is only seen after severe challenges such as massive fungal infection (Krekling et al. 2000, 2004; Krokene et al. 2003). Production of extra PP cells may also be induced by treatment with methyl jasmonate (Krokene et al. 2007), an elicitor of plant defense responses that induces similar responses as fungal pathogens and insects in conifers (Franceschi et al. 2002; Martin et al. 2002; Hudgins et al. 2004; Krokene et al. 2008). With time, extra PP cells appear several centimeters away from the point of induction and can be quite abundant, covering 50% of the phloem circumference in Norway spruce 15 weeks after a massive fungal infection (Krokene et al. 2003). Because differentiation of extra PP cells is a relatively slow process compared to the activation of existing ones, it probably enhances long-term resistance rather than resistance to an on-going attack. Production of extra PP cells seems to be involved in the phenomenon of systemic induced resistance in Norway spruce (Krokene et al. 2003). PP cells are also instrumental in the process of wound periderm formation (Franceschi et al. 2000). This is an essential part of conifer defenses, as it serves to wall off damaged regions and re-establish a continuous surface barrier (see also Ginzberg this volume). Wound periderms are produced by activation of existing PP cells, which begin to divide to form a wound phellogen or cork cambium (Fig. 7.3B). Similar to the normal periderm this meristem produces cork tissue (phellem) outwards and phelloderm inwards, and thus regenerates a continuous surface barrier (Franceschi et al. 2005). Damaged tissues outside this new periderm are isolated

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from nutrient supplies through the secondary phloem and will eventually be shed as new phloem is produced by the vascular cambium.

7.2.3 Evidence for Defensive Roles of PP Cells The anatomical organization, chemical nature and inducible responses of PP cells suggest that they are important in resistance against attacking organisms. A defensive role for PP cells is supported by inoculation experiments with bark beetleassociated bluestain fungi, showing that tree resistance developed over a time frame that paralleled the time required to activate PP cells (Krokene et al. 2003). Trees pretreated with a sublethal fungal infection three to nine weeks before a massive artificial infection showed strong activation of PP cells, and were much more resistant to the subsequent challenge than untreated trees (Krokene et al. 1999, 2003). Trees pretreated one week before mass-infection showed no activation of PP cells and were as susceptible as untreated control trees (Krokene et al. 2003). In this experiment, the constitutive PP cell barrier was circumvented, as fungus was artificially inoculated into wounds going all the way to the vascular cambium. When fungal inoculations are made into shallow bark wounds, ending in the middle of the PP cell layers, fungal growth is completely restricted, confirming a role in defense also for the constitutive, concentric layers of PP cells (Franceschi et al. 2000). Many bluestain fungi are successful conifer pathogens because they circumvent bark defenses by hitching a ride with scolytid bark beetles tunneling through the bark to the vascular cambium (e.g., Paine et al. 1997; Franceschi et al. 2000, 2005). The exact chemical nature of the phenolic material inside the PP cells is not known. The conclusion that the material is polyphenolic in nature is based on microscopical evidence, such as structural similarity to polyphenolics in other plant cell types, autofluorescence, staining properties, and localization of PAL to the plasma membrane and cytoplasm of PP cells by immunolocalization (Franceschi et al. 1998). Other important enzymes involved in phenol synthesis, such as chalcon and stilbene synthases, have also been found to increase in concentration in conifer bark after fungal infection, emphasizing the role of phenolics in bark defenses (Brignolas et al. 1995a; Richard et al. 2000; Nagy et al. 2004). There are also numerous direct chemical analyses of conifer phloem showing high concentrations of soluble phenolics, such as flavonoids and stilbenes (Lindberg et al. 1992; Lieutier et al. 1991, 1996, 2003; Brignolas et al. 1995a, b, 1998; Dixon and Paiva 1995; Bois and Lieutier 1997; Evensen et al. 2000; Viiri et al. 2001; Zeneli et al. 2006). However, the soluble phenolics that lend themselves to routine chemical analysis do not appear to be crucial for defense, as they were only weakly inducible by fungal inoculation, mechanical wounding, and bark beetle attack (Erbilgin et al. 2006; Zeneli et al. 2006). PP cell activation may thus be related to changes in more complex phenolics, such as high molecular weight condensed tannins or cell wall-bound phenolics, but this remains to be tested.


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7.3 Traumatic Resin Ducts 7.3.1 Resin-Based Defenses in Conifers Conifer resin is composed largely of terpenes, made up chiefly of monoterpenes (C10 ) and diterpenes (C20 ), with small amounts of sesquiterpenes (C15 ) and other compounds (Gershenzon and Croteau 1991; Bohlmann this volume). Resin is generally considered to be important in conifer defense because of its physical properties and its toxic and repellent effects on many herbivores and pathogens (Reid et al. 1967; Shrimpton and Whitney 1968; Bordasch and Berryman 1977; Raffa et al. 1985; Gijzen et al. 1993; Bohlmann this volume). Conifer resin is synthesized and accumulated in specialized secretory structures of different anatomical complexity, ranging from scattered individual resin cells and sac-like resin cavities lined with epithelial cells, to complex networks of interconnected radial and axial resin ducts. These resin structures may be present constitutively or they may be inducible, and they are found in many different tissues, including xylem, phloem, cortex and needles. Resin producing structures are found in many conifer lineages, but they are not uniformly important. In fact, most conifers in the families Araucariaceae, Podocarpaceae, Cupressaceae, Cephalotaxaceae, and Taxaceae (the non-Pinaceae families) lack preformed resin structures in phloem and xylem (Bannan 1936; Fahn 1979; Wu and Hu 1997), and inducible resin structures only appear in some genera (Hudgins et al. 2004; Franceschi et al. 2005; Table 7.1). Instead, the nonPinaceae species seem to rely mainly on massive constitutive bark defenses in the form of PP cells and lignified fiber cells (see Section 7.2.1). It is a striking and almost paradoxical fact that the Pinaceae conifers that are relying on resin-based defenses are more susceptible to tree-killing bark beetles (family Scolytidae) as compared to species in the non-Pinaceae group which rarely suffer from bark beetle attacks (Hudgins et al. 2004; Franceschi et al. 2005). The specialization of treekilling bark beetles on resin-producing conifers includes the use of resin as a precursor for production of population-aggregating pheromones (e.g., Vanderwel and Oehlschlager 1987). Effective aggregation pheromones are probably essential for tree-killing, since small herbivores like bark beetles must attack in large numbers in order to overwhelm and kill large host trees (Franceschi et al. 2005). Bark beetles are thus using one of the tree’s primary chemical defenses to their advantage, illustrating how co-evolution between insect herbivores and host trees can turn a defense into a weakness. Still, since most conifers, both Pinaceae and non-Pinaceae species, usually are well-defended against bark beetles, these contrasting strategies may also serve as an example of how completely different defense strategies can be effective against the same pest (Franceschi et al. 2005). Constitutive resin structures are present in the cortex and phloem of most Pinaceae and a few non-Pinaceae conifers, usually in the form of resin ducts or resin cavities (Fig. 7.5; Table 7.1). In addition, many Pinaceae species have scattered constitutive resin ducts in normal xylem (Table 7.1). The axial resin ducts in the cortex (the

Cortical ducts

yes yes no yes yes yes no yes yes yes yes

Species

>40 2–4 1 9 1 3 14 5 1 35 115

19 21 1

Genus

Abies Cedrus Pseudolarix Tsuga Nothotsuga Keteleeria Larix Pseudotsuga Cathaya Picea Pinus

Araucaria Agathis Wollemia

no? no? no? no? no? no? no? no? no? no? no? axial no? no?

(no)3 no no?

Inducible

cavities cavities cavities cavities radial radial radial radial radial radial radial

Phloem Constitutive

no no no

no no no no no axial network1 network network network network

Xylem Constitutive

no no? no?

cavities axial cavities cavities axial axial? axial axial axial? axial (axial)2

Inducible

Hudgins et al. (2004) Penhallow (1907) Heady et al. (2002)

Hudgins et al. (2004) and Wu and Hu (1997) Hudgins et al. (2004) and Wu and Hu (1997) Penhallow (1907), Bannan 1936 and Wu and Hu (1997) Hudgins et al. (2004) and Wu and Hu (1997) Wu and Hu (1997) Wu and Hu (1997) Hudgins et al. (2003a) and Wu and Hu (1997) Hudgins et al. (2004) and Wu and Hu (1997) Wu and Hu (1997) Hudgins et al. (2004) and Wu and Hu (1997) Hudgins et al. (2004) and Wu and Hu (1997)

Reference

Table 7.1 Different types of resin structures in conifer stems. The genera are grouped by family, starting with the Pinaceae (showing all 11 genera), continuing with Araucariaceae (1 of 3 genera), Podocarpaceae (1 of 18–19 genera), Cupressaceae (7 of 33 genera), Cephalotaxaceae (1 of 3 genera), and Taxaceae (2 of 3 genera)

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5 6 1 1 1 1 >16 >117

5–6 16

9 2

Thuja Chamaecyparis Sequoia Sequoiadendron Metasequoia Cryptomeria Cupressus + 26 genera

Torreya +2 genera

Taxus +2 genera

3

2

Cortical ducts

no

no

cells no radial? radial? radial? no (no)3

no

Phloem Constitutive

no

no

no? axial no no no axial axial

no

Inducible

no

no

cells? no no no no no no

no

Xylem Constitutive

Network = combination of radial and axial resin ducts Weak induction of resin ducts that are restricted to the immediate vicinity of the point of induction Non-functional bundles of epithelial cells are produced constitutively and activated in response to damage/attack

105 >65

Podocarpus + 17–18 genera

1

Species

Genus


no

no

no? no axial axial axial no no

no

Inducible

Hudgins et al. (2003a)

Penhallow (1907)

Hudgins et al. (2003b) Yamada et al. (2002) Hudgins et al. (2004) Hudgins et al. (2004) Hudgins et al. (2004) Hudgins et al. (2004) Hudgins et al. (2004)

Hudgins et al. (2004)

Reference

7 Induced Anatomical Defenses in Conifers 159

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outer part of the young stem bark) may be very large compared to other resin ducts (Figs. 7.2A and 7.5A). They are formed in a circumferential ring as the cortex is produced during primary stem development, and may be present for at least 25 years in Norway spruce (Christiansen et al. 1999a). The cortex is an important defense barrier in the early stages of stem growth, but its defensive roles are gradually taken over by the secondary phloem as this develops more extensive layers (Franceschi et al. 2005). The constitutive resin ducts in the phloem are always radial, and reside within multiseriate radial rays surrounded by ray parenchyma cells. The crosssection of these ducts increases in size in the older parts of the bark and can be quite extensive (Christiansen et al. 1999a; Fig. 7.3A). Radial resin ducts occur primarily in the phloem, but some run continuously from the phloem to the sapwood, where they are interconnected with the axial resin ducts (Fig. 7.5E; Christiansen et al. 1999a; Nagy et al. 2000). A complex constitutive resin duct network with interconnected radial and axial ducts in phloem and xylem are found only in some Pinaceae genera (Pinus, Picea, Larix, Pseudotsuga, Keteleeria, Cathaya; Table 7.1), and is most highly developed in the genus Pinus which seems to rely heavily on constitutive resin (Bannan 1936; Vité 1961; Johnson and Croteau 1987). Resin ducts are lined with plastid-enriched epithelial cells that produce and secrete resin into an extracellular lumen, where it is stored under pressure (Charon et al. 1987; Gershenzon and Croteau 1990; Nagy et al. 2000; Fig. 7.6A). The simpler resin cells accumulate resin internally under pressure and may expand into quite large structures (Fig. 7.5B). Resin ducts form schizogenously as the epithelial cells pull apart during resin duct formation (Nagy et al. 2000). The epithelial cells lining radial and axial resin ducts are thin-walled and long-lived, in contrast to the epithelial cells of the resin cavities, which are short-lived and gradually become lignified during development (Bannan 1936; Fahn 1979).

7.3.2 Anatomy and Development of Traumatic Resin Ducts In addition to constitutive resin structures many conifers develop inducible resin structures in response to biotic and abiotic challenges, such as insect attack, pathogen infection, frost damage, or physical wounding (e.g., Bannan 1936; Alfaro 1995; Kytö et al. 1996; Hudgins et al. 2003a, 2004). In the Pinaceae, inducible resin structures usually occur as so-called pathological or traumatic axial resin ducts in the xylem, but axial resin ducts are also induced in the phloem of a few non-Pinaceae genera (Table 7.1). The nature of the vascular cambium implies that inducible resin ducts must be axial and not radial. The cambium can relatively quickly be re-programmed to produce functional axial resin ducts that may be several meters long (e.g., Christiansen et al. 1999b; Nagy et al. 2000), whereas it would require years to produce radial ducts of any extent. TRDs form in tangential bands in the xylem of many Pinaceae species (Figs. 7.3B and 7.5D). These bands are often surrounded by small tracheids with thickened cell walls and are visible to the naked eye as a punctuated line within the annual ring


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Fig. 7.5 Cross-sections of resin-containing structures in conifers. (A) An axially oriented resin duct in the outer cortical region of the phloem of balsam fir (Abies balsamea). Bar, 50 ␮m. The circular cortical resin ducts are lined with small epithelial cells which are surrounded by sheet cells containing phenolic material. (B) In grand fir (Abies grandis) resin is produced and stored in individual resin cells in the secondary phloem. Bar, 50 ␮m. (C) Low magnification of Norway spruce (Picea abies) bark and sapwood, showing traumatic resin ducts within the sapwood 3 weeks after inoculation with the blue-stain fungus Ceratocystis polonica. The traumatic resin ducts are visible as small, tangentially oriented channels close to the cambium. (D) Closer look at traumatic resin ducts in the xylem of Norway spruce after fungal inoculation. The traumatic resin ducts are characteristically associated with radial rays. (E) Cross-section demonstrating luminal continuity between several coalescing traumatic resin ducts and a radial resin duct traversing the phloem and xylem. Bars, 50 ␮m

(false annual rings; Fig. 7.5C). The formation of functional TRDs with secretory activity is quite rapid, and requires two to four weeks in Norway spruce (Nagy et al. 2000). TRDs develop in the same way as normal axial resin ducts, by schizogenesis between incipient epithelial cells when these are still close to the cambial zone (Fig. 7.6B). Sometimes large transverse resin ducts can be produced by tangential anastomosis of neighboring ducts (Gerry 1942; Fig. 7.5E). If the inducing

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Fig. 7.6 Cross-sections of the epithelial cells of developing traumatic resin ducts. (A) In Norway spruce, the epithelial cells lining the resin duct are characterized by having dense cytoplasm, with many plastids and vacuoles, and conspicuously enlarged nuclei. The numerous plastids are the primary site for resin synthesis. Bar, 10 ␮m. (B) Early in their development the traumatic resin ducts are often surrounded by living parenchyma cells full of starch grains and phenolic material. Bar, 25 ␮m

stimulus is strong, an almost complete tangential ring of TRDs can be produced, and with time, the ducts may extend several meters from the point of induction in spruce (Christiansen et al. 1999b). In other genera, such as Abies, Tsuga, Cedrus and Pseudolarix, TRDs mainly form in the vicinity of wounds (Bannan 1936; Wu and Hu 1997).


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The TRD system may represent a considerable volume, and contribute significantly to a tree’s overall resin production. After massive induction, TRDs are much more abundant than normal xylem ducts, and may extend over 50%–100% of the xylem circumference in Norway spruce (Franceschi et al. 2002; Krokene et al. 2003). TRDs are also much larger than constitutive xylem ducts; in Norway spruce for example, they are often twice as wide and represent a volume that is four times greater per unit duct length. TRDs form an interconnected network of resinfilled cavities tangentially in the new sapwood, as well as radially with luminal continuity between adjacent TRDs and radial constitutive resin ducts (Fig. 7.5E). This organization facilitates resin flow both axially along the trunk, as well as radially towards the bark surface (Nagy et al. 2000; Krokene et al. 2003). TRDs appear to be derived from xylem mother cells (Werker and Fahn 1969; Nagy et al. 2000). The strict distribution pattern of TRDs in tangential bands implies a combination of predisposition and a tangential spread of a stimulus resulting in coordinated induction and differentiation of the xylem mother cells. With increasing distance from the point of induction, TRDs are gradually reduced in size and number, and are found closer and closer to the cambium (Krekling et al. 2004). There thus appears to be a time-lag in the production and differentiation of the TRDs away from the stimulus, which would be consistent with a signal propagating a developmental wave traveling in the axial direction at about 2.5 cm per day (Krekling et al. 2004). TRD formation appears to be activated through the octadecanoid pathway, involving jasmonate and ethylene signaling (Franceschi et al. 2002; Martin et al. 2002; Hudgins et al. 2003a, 2004, 2006; Hudgins and Franceschi 2004; Miller et al. 2005; Ralph et al. 2007).

7.3.3 Defensive Roles of TRDs TRD formation in Norway spruce is part of a complex defense response that is rapid and coordinated, resulting in an extensive resin-secreting network. The result is massive accumulation of resin and enhanced resin flow at the site of attack (Phillips and Croteau 1999; Nagy et al. 2000). Artificial inoculation or wounding, leading to TRD formation and increased synthesis of resin, can thus be used as a technique to improve and prime a tree’s resistance against insects and pathogens. For example, Norway spruce trees that were pretreated with a sublethal fungal infection developed abundant TRDs which rendered the trees much more resistant to a subsequent massive infection after three to nine weeks (Krokene et al. 1999, 2003). The ability of a tree to undergo traumatic resinosis can be used to screen for resistance (Krokene et al. 2008). For example, resistant clones of Norway spruce and white spruce (Picea glauca) produce more TRDs more quickly as compared to susceptible clones (Tomlin et al. 1998; Nagy et al. 2000; Krekling et al. 2004). In white spruce, large inner and dense outer resin canals were correlated with resistance against the white pine weevil, Pissodes strobi (Peck) (Alfaro et al. 2004). There is ample evidence that conifer resin is effective in defense against insects. In several conifers, the concentration of monoterpenes is enhanced in tissues

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attacked by bark beetles and their associated blue-stain fungi (Russell and Berryman 1976; Cates and Alexander 1982; Raffa and Berryman 1982, 1983; Paine et al. 1987; Lewinsohn et al. 1991). Tomlin et al. (2000) found that simulated white pine weevil damage enhanced the monoterpene to resin acid ratio in stem regions distant from the wounded site in resistant trees, but only slightly in susceptible trees. Monoterpene-enriched resin, which is less viscous than constitutive resin, probably flows more readily into oviposition cavities and larval mines, thus being more effective in inhibiting/killing immature weevils. Resin may also inhibit invading insects and mycelial growth of fungal pathogens due to its toxic properties. For example, Klepzig et al. (1995) found that monoterpenes in red pine resin inhibit fungal germination and mycelial growth, as well as beetle tunneling. Alpha-pinene in particular, the most abundant monoterpene in red pine phloem, exhibited strong biological activity towards insects and fungi (Klepzig et al. 1995). Resin diterpenes are considered to be insecticidal and fungicidal, and are important in wound sealing. An open wound is a potential infection pathway for attacking insects and pathogenic fungi, and effective wound sealing is decisive in preventing invasions. The diterpene abietic acid is important for oxidative polymerization of resin and dehydroabietic acid for resin viscosity and crystallization (Phillips and Croteau 1999). Resistant white spruce trees appear to produce more volatile terpenes and diterpene resin acids at the area of wounding than susceptible trees (Tomlin et al. 2000). The induction of functional TRDs is usually too slow to be effective against rapid mass-attacks by bark beetles, which may be completed in a few days (Paine et al. 1997). However, TRD formation may contribute to defense when bark beetle attacks are slowed down by e.g. unfavorable weather conditions (Franceschi et al. 2000), and may also be important in protecting the trees against opportunistic wound colonizers after e.g. mechanical damage to the bark.

7.4 Perspectives Invaders attempting to colonize healthy conifers have to struggle their way through multiple layers of periderm, PP cells and/or fiber cells. However, the ray cells running axially through the secondary phloem and xylem represent a potential weak point in this defense barrier. Since the rays are the only tissue type in the stem that is oriented radially they form a major direct link between phloem and sapwood. This potential loophole must therefore be expected to be well-defended, and immunolabeling studies in Norway spruce have indeed demonstrated that rays are a major site for PAL activity (Franceschi et al. 1998), and probably other defensive elements as well. Anatomical studies in Scots pine suggest that pathogen infection induces changes in ray cells, including increase in cell size and a more active cytoplasm that may be related to increased cellular activity and resin production (Nagy et al. 2006). However, as far as we know these preliminary studies are the only ones looking at inducible defenses in ray cells, and there are no detailed studies of ray


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cell structure and function. The rays are not only involved in defending the bark and outer sapwood, but are probably also instrumental in heartwood formation and defense against e.g. wood rotting fungi, since they are the only route for translocation of substances between the living phloem and the inner sapwood (Shain 1967; Kwon et al. 2001; Franceschi et al. 2005). With their radial orientation, the rays are also expected to play important roles in signaling and transport of chemicals between phloem and xylem. Chemical and molecular characterization of the central cell types in conifer defense, such as radial rays, PP cells, and resin duct epithelial cells, is strongly needed. The data available so far represent whole tissues (e.g., the whole secondary phloem), and the results are thus an average of information of different tissue and cell types. Recent technological and analytical developments have made it possible to dissect specific cell types and characterize their content using both chemical and molecular techniques. Laser microdissection enables effective isolation of single cells and is widely used in the biomedical field (Emmert-Buck et al. 1996; Day et al. 2005). More recently, laser microdissection has been adapted for use with many plant species, including Arabidopsis, rice, maize and others (Ohtsu et al. 2007). The only example from conifers so far is the dissection and chemical analysis of stone cells in Norway spruce (Li et al. 2007). Using laser microdissection in combination with sensitive instrumental analysis such as mass spectroscopy and cryogenic NMR-spectroscopy, it will be possible to chemically characterize pure cell populations. This represents a new approach for the cell-specific localization of lowmolecular weight compounds in plants, and has been termed micro-phytochemistry (Li et al. 2007). In addition to metabolites, proteins, DNA, and RNA can be analyzed from microdissected samples. This powerful tool can thus give us detailed information about the chemical and molecular nature of all the important cell types in conifer defense.

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insect-induced expression, and cellular and subcellular localization in spruce and Douglas fir. Plant Physiol 143:410–424 Reglinski T, Stavely FJL, Taylor JT (1998) Induction of phenylalanine ammonia lyase activity and control of Sphaeropsis sapinea infection in Pinus radiata by 5-chlorosalicylic acid. Eur J For Pathol 28:153–158 Reid RW, Whitney HS, Watson JA (1967) Reactions of lodgepole pine to attack by Dendroctonus ponderosae Hopkins and blue stain fungus. Can J Bot 45:1115–1126 Richard S, Lapointe G, Rutledge G, Séguin A (2000) Induction of chalcone synthase expression in white spruce by wounding and jasmonate. Plant Cell Physiol 41:982–987 Ruel JJ, Ayres MP, Lorio PL (1998) Loblolly pine responds to mechanical wounding with increased resin flow. Can J For Res 28:596–602 Russell CE, Berryman AA (1976) Host resistance to the fir engraver beetle. 1. Monoterpene composition of Abies grandis pitch blisters and fungus-infected wounds. Can J Bot 54:14–18 Schmidt A, Zeneli G, Hietala AM, Fossdal CG, Krokene P, Christiansen E, Gershenzon J (2005) Induced chemical defenses in conifers: biochemical and molecular approaches to studying their function. In: Romeo J (ed) Chemical ecology and phytochemistry of forests and forest ecosytems. Elsevier, Amsterdam, pp 1–28 Shain L (1967) Resistance of sapwood in stems of loblolly pine to infection by Fomes annosus. Phytopathology 57:1034–1045 Shrimpton DM, Whitney HS (1968) Inhibition of growth of blue stain fungi by wood extractives. Can J Bot 46:757–761 Tomlin ES, Alfaro RI, Borden JH, He FL (1998) Histological response of resistant and susceptible white spruce to simulated white pine weevil damage. Tree Physiol 18: 21–28 Tomlin ES, Antonejevic E, Alfaro RI, Borden JH (2000) Changes in volatile terpene and diterpene resin acid composition of resistant and susceptible white spruce leaders exposed to simulated white pine weevil damage. Tree Physiol 20:1087–1095 Vanderwel D, Oehlschlager AC (1987) Biosynthesis of pheromones and endocrine regulation of pheromone production in Coleoptera. In: Prestwich GD, Blomquist GJ (eds) Pheromone biochemistry. Academic Press, New York, pp 175–215 Vité JP (1961) The influence of water supply on oleoresin exudation pressure and resistance to bark beetle attack in Pinus ponderosa. Contributions from the Boyce Thompson Institute 21:37–66 Viiri H, Annila E, Kitunen V, Niemelä P (2001) Induced responses in stilbenes and terpenes in fertilized Norway spruce after inoculation with blue-stain fungus, Ceratocystis polonica. Trees 15:112–122 Wainhouse D, Cross DJ, Howell RS (1990) The role of lignin as a defense against the spruce bark beetle Dendroctonus micans – effect on larvae and adults. Oecologia 85:257–265 Wainhouse D, Rose DR, Peace AJ (1997) The influence of preformed defences on the dynamic wound response in spruce bark. Funct Ecol 11:564–572 Werker E, Fahn A (1969) Resin ducts of Pinus halepensis Mill.—their structure, development and pattern of arrangement. Bot J Linn Soc 62:379–411 Wu H, Hu ZH (1997) Comparative anatomy of resin ducts of the Pinaceae. Trees Struct Funct 11:135–143 Zeneli G, Krokene P, Christiansen E, Krekling T, Gershenzon J (2006) Methyl jasmonate treatment of large Norway spruce (Picea abies) trees increases the accumulation of terpenoid resin components and protects against infection by Ceratocystis polonica, a bark beetle-associated fungus. Tree Physiol 26:977–988

Part B

Production of Secondary Metabolites

Chapter 8

Insect-Induced Terpenoid Defenses in Spruce Jörg Bohlmann

Conifers produce an array of hundreds of different terpenoids as part of their complex chemical, physical, and ecological defenses against insect pests and pathogens. Terpenoid chemicals exist both as constitutive and as massively induced defenses in conifers. It is thought that the diversity of terpenoid chemicals serves as a multilayered chemical shield in long-lived conifer trees that provides a lasting protection against the much faster evolving insect pests and potential pathogens. The formation of terpenoid defenses in conifers involves the activity of two pathways, the methylerythritol phosphate pathway and the mevalonate pathway, which lead to the five-carbon precursors of terpenoid biosynthesis. The many monoterpenoids, sesquiterpenoids, and diterpene resin acids, which are present in oleoresin mixtures, are then formed by families of enzymes belonging to the classes of prenyl transferases, terpenoid synthases, and cytochrome P450 dependent monooxygenases. The genes for almost all the enzymatic steps in terpenoid oleoresin biosynthesis have been identified in species of spruce, which have thus been established as a conifer reference system to study constitutive and induced terpenoid defenses using biochemical, molecular genetic, and genomic and proteomic approaches. At the histological and cellular levels, oleoresin terpenoids are produced and accumulate constitutively in large quantities in specialized anatomical structures that are found in most organs and tissues. In many conifer species, biosynthesis and accumulation of terpenoids is further enhanced as part of the induced defense in response to insect attack or fungal infection. In this chapter, I will discuss selected aspects of terpenoid defenses in conifers against insects and pathogens.

8.1 Introduction Conifers (i.e., members of the pine family; Pinaceae) include some of the longestliving and tallest organisms on earth. Their tall physical structure and long lifespan

J. Bohlmann Michael Smith Laboratories, University of British Columbia, Vancouver, B.C., Canada e-mail: [email protected]


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make them prominent targets in space and time for many potential herbivores and pathogens. The successful defense and resistance of conifers against most herbivores and pathogens can be explained, at least in part, by the formation of a diverse array of monoterpenoid, sesquiterpenoid, and diterpene resin acid defense chemicals. These compounds accumulate in large amounts in form of preformed or induced oleoresin mixtures. Some terpenoids, in particular monoterpenes and sesquiterpenes, can also be actively emitted as volatile organic compounds with functions as semiochemicals from conifer needles. Conifer oleoresin is stored in specialized anatomical structures such as resin canals, resin blisters, or resin cells in stems, roots, or needles. The developmental programs for the constitutive formation of these specialized, resinaccumulating structures are not understood; except, it is now well established that the formation of so-called traumatic resin ducts is induced in the cambium zone of conifer stems in response to insect or fungal attack, by mechanical wounding, or by chemical elicitation with methyl jasmomate (MeJA) or ethylene treatment (Krokene et al. this volume). In recent years, several reviews have been published on the topic of terpenoid oleoresin defenses in conifers (e.g., Bohlmann and Croteau 1999; Phillips and Croteau 1999; Trapp and Croteau 2001a; Keeling and Bohlmann 2006a, b). The families of terpenoid synthases (TPS) and cytochrome P450 dependent monooxygenases (P450) play a central role in the formation of terpenoid chemical diversity and for phenotypic plasticity in conifer defense (Bohlmann et al. 1998a; Martin et al. 2004; Ro et al. 2005). Both, the TPS and P450 are encoded in multigene families in conifers (Martin et al. 2004; Hamberger and Bohlmann 2006). These gene families are thought to contribute, on the genomic, molecular, and biochemical levels, much to the diversity and plasticity of constitutive and induced terpenoid defenses in the long-lived conifers. In contrast to the TPS and P450, we know relatively little about the early steps of terpenoid biosynthesis in conifers with only a few studies published on prenyltransferases (PT; Tholl et al. 2001; Burke and Croteau 2002; Martin et al. 2002; Schmidt and Gershenzon 2007) or earlier steps in conifer isoprenoid biosynthesis (Phillips et al. 2007). However, a recent large-scale spruce genomics project has identified genes (i.e., expressed sequences tags, ESTs; and full-length cDNAs, FL-cDNAs) for almost every single step in conifer monoterpenoid, sesquiterpenoid, and diterpenoid biosynthesis (Ralph et al. 2006; Ralph and Bohlmann, in preparation). In building on pioneering work by Clarence (Bud) Ryan and coworkers, the use of MeJA as an elicitor to induce plant defenses enabled us and others to develop a detailed characterization of inducible terpenoid defenses in several conifer species such as Norway spruce (Picea abies), Sitka spruce (P. sitchensis), White spruce (P. glauca) and Douglas fir (Pseudotsuga menziesii; e.g., Franceschi et al. 2002; Martin et al. 2002, 2003a; Fäldt et al. 2003; Hudgins et al. 2003; Huber et al. 2005a, b; Miller et al. 2005; Ralph et al. 2006; Erbilgin et al. 2006; Zeneli et al. 2006; Phillips et al. 2007). By treating conifer seedlings, mature trees, or even cell cultures with MeJA, it became possible to accurately measure the quantitative and qualitative changes of traumatic resin terpenoids, detect the dynamics of enzyme activities and transcript levels induced in the traumatic terpenoid defense response, and analyze

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the induced and active emission of terpenoid volatiles. In addition, the non-invasive treatment with MeJA, as opposed to mechanical wounding or insect attack, allowed for the analysis of temporal and spatial patterns of cell differentiation during traumatic resin duct formation in the cambium zone of spruce stems. Application of MeJA provided a powerful tool to mimic at least some of the effects of insect feeding in spruce, as has been shown in the defense response of Sitka spruce to white pine weevil (Pissodes strobi; Miller et al. 2005). Beyond the use of MeJA as an exogenous elicitor, both ethylene and octadecanoid signaling appear to be involved in the endogenous signaling of the induced traumatic resin response in spruce and Douglas fir (Hudgins and Franceschi 2004; Miller et al. 2005; Hudgins et al. 2006; Ralph et al. 2007). The MeJA- or insect-induced accumulation of terpenoids in traumatic resin ducts in stems and roots, as well as the induced emission of terpenoid volatiles from needles, is controlled at least in part by up-regulation of terpenoid biosynthesis (Martin et al. 2002, 2003a; Huber et al. 2005b; Miller et al. 2005; Keeling and Bohlmann, 2006a). In this process, transcript levels and/or enzyme activities for several steps in terpenoid formation are up-regulated in the methylerythritol phosphate (MEP) pathway, in the subsequent prenyltransferase steps, and in the late steps catalyzed by TPS and P450s. The analysis of more than 200,000 spruce ESTs and 6,464 high-quality finished FLcDNAs along with transcriptome and proteome analyses have identified many other insect- and MeJA-regulated processes in the induced chemical defense and resistance of Sitka spruce against white pine weevil and spruce budworms (Choristoneura occidentalis; Ralph et al. 2006, 2007b; Lippert et al. 2007; Ralph and Bohlmann, in preparation).

8.2 Biochemistry of Terpenoid Biosynthesis in Conifer Defense The progress in research on the biochemistry and molecular biology of terpenoids in conifer defense has recently been reviewed (Martin and Bohlmann 2005; Keeling and Bohlmann 2006a, b; Phillips et al. 2006) and a number of earlier reviews on this topic are available (e.g., Bohlmann and Croteau 1999; Phillips and Croteau 1999; Trapp and Croteau 2001a). Terpenoids represent the largest group of known plant secondary metabolites. The three large classes of monoterpenoids, sesquiterpenoids, and diterpene resin acids are the most abundant terpenoid defense chemicals in conifers. In particular, the monoterpenoids and diterpene resin acids constitute much of the conifer oleoresin volume. In addition, sesquiterpenoids contribute substantially to the structural diversity of chemicals in the oleoresin mixture. The biosynthesis of monoterpenoids, sesquiterpenoids, and diterpene resin acids begins with the formation of the five carbon building blocks, isopentenyl diphosphate (IDP) and its isomer, dimethylallyl diphosphate (DMAPP). In plants, two pathways exist for the formation of these precursors, the mevalonic acid (MEV) pathway and the MEP pathway. Based on large-scale EST and FLcDNA sequencing, all but one gene for the MEV and MEP pathways have been cloned from Sitka spruce and/or white

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spruce (Ralph et al. 2006; Ralph and Bohlmann, in preparation). These genomics efforts in Sitka spruce and white spruce, by extension of gene discovery to other conifer species, led to the identification and functional characterization of a small family of differentially MeJA- and fungal-induced genes for the first step of the MEP pathway, deoxyxylulose phosphate synthase (DXPS), in Norway spruce (Phillips et al. 2007). Based on our studies with Norway spruce DXPS in cell suspension cultures, it appears that the different isoforms of DXPS may have specific functions in regulation of substrate flux in primary isoprenoid metabolism and secondary terpenoid defense metabolism. Following the formation of IDP and DMAPP, a group of PTs catalyze 1’-4 condensation reactions coupling IPP with an allylic prenyl diphosphate. Specifically, geranyl diphosphate (GDP) synthase forms the C10 precursor of monoterpenoids, farnesyl diphosphate (FDP) synthase forms the C15 precursor of sesquiterpenoids, and geranylgeranyl diphosphate (GGDP) synthase produces the C20 precursor for diterpenoids. A few conifer PTs have been characterized in grand fir (Abies grandis; Tholl et al. 2001; Burke and Croteau 2002) and more recently in Norway spruce (Schmidt and Gershenzon 2007) and a large number of distinct PTs have been identified with ESTs and FLcDNAs in Sitka spruce and white spruce (Ralph and Bohlmann, in preparation). Although it is thought that PTs may control flux of pathway intermediates at branch points of monoterpenoid, sesquiterpenoid, and diterpenoid biosynthesis in the induced defense of conifers, except for a few studies showing up-regulation of some PT activities and transcript levels, this aspect of the regulation of terpenoid biosynthesis is not well understood and will require much further characterization (Martin et al. 2002; Schmidt and Gershenzon 2007; Ralph and Bohlmann, in preparation). TPS utilize the three prenyl diphosphates GDP, FDP, and GGDP as substrates in the formation of the hundreds of structurally diverse monoterpenoids, sesquiterpenoids, and diterpenoids. The TPS exist in large gene families and are arguably the best characterized genes and enzymes of chemical defenses in conifers (Bohlmann et al. 1998a; Martin et al. 2004; Keeling and Bohlmann 2006a). Much of the work on TPS in induced conifer defense is based on the early and pioneering studies by Rodney Croteau and coworkers at Washington State University. The TPS employ an electrophilic reaction mechanism, assisted by divalent metal ion cofactors (Davis and Croteau 2000). The prenyl diphosphate substrates are ionized or protonated by TPS to produce reactive carbocation intermediates. The carbocation intermediates can then rearrange within the spatial constraints of the TPS active site and are eventually quenched to yield the many different cyclic and acyclic terpenoid products (Starks et al. 1997; Cane 1999; Wise and Croteau 1999; Christianson 2006). While some TPS form only a single product, the majority of TPS characterized to date, including many TPS identified in conifers, produce arrays of multiple products from a single substrate (Steele et al. 1998; Fäldt et al. 2003; Martin et al. 2004). Based on their substrate specificities, the single- and multi-product TPS are grouped into classes of mono-TPS, sesqui-TPS and di-TPS, which are responsible for the formation of the many simple (acyclic or single ring structure) and more intricate (two or more ring structures) terpene skeletons of conifer mono-, sesqui-, and diterpenoids. Many of the known conifer TPS appear to exert tight control over the stereochemistry of


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products formed and usually one enantiomer dominates any given TPS product profile. To date, approximately 50 different conifer TPS have been cloned and many of them have been functionally characterized in Norway spruce, Sitka spruce and white spruce (Byun McKay et al. 2003, 2006; Fäldt et al. 2003; Martin et al. 2004; Keeling et al. 2006a; Ralph and Bohlmann, in preparation). In addition, TPS have been cloned and characterized from grand fir (e.g., Stofer Vogel et al. 1996; Bohlmann et al. 1997, 1998a, 1999; Steele et al. 1998), loblolly pine (Phillips et al. 2003; Ro and Bohlmann 2006) and Douglas fir (Huber et al. 2005a). While the majority of terpenoid products formed by the conifer mono-TPS and sesqui-TPS accumulate in the oleoresin without any further apparent biochemical modifications, the diterpene olefins produced by the di-TPS may be subject to oxidations catalyzed by P450s (Keeling and Bohlmann 2006b). To date only a single P450 in the formation of diterpene resin acids has been cloned and functionally characterized (Ro et al. 2005). The loblolly pine PtAO P450 enzyme, abietadienol/abietadienal oxidase, is a multi-substrate and multi-functional diterpene oxidase in the CYP720 family of plant P450 enzymes. PtAO catalyzes at least two of the three consecutive oxidation steps in the formation of conifer diterpene resin acids and the enzyme efficiently uses several different diterpene alcohols and diterpene aldehydes as substrates. Through the combination of accepting multiple diterpenoid substrates and by catalyzing at least two consecutive oxidations, this P450 enzyme leads to the formation of several different diterpene resin acids found in conifer oleoresin. The ability of PtAO to catalyze consecutive oxidation steps is similar to the activity of angiosperm P450s in the biosynthesis of the diterpene gibberellin phytohormones (Ro et al. 2005; Keeling and Bohlmann 2006b). A further analysis of the nearly half a million conifer ESTs from spruce and loblolly pine revealed a large number of new P450s in the CYP85 clan that may be involved in terpenoid oxidation in conifers (Hamberger and Bohlmann 2006). While conifer P450s putatively involved in terpenoid phytohormone formation, such as gibberellic acid biosynthesis, appear to be expressed as single-copy genes in Sitka spruce and loblolly pine, we found substantial expansion of conifer-specific P450 subfamilies that are likely associated with terpenoid secondary metabolism and defense. Since conifer P450 enzymes are extremely difficult to study as native enzymes in crude or cell-free extracts from bark, needle, or wood tissues, it was necessary to develop an efficient recombinant expression and assay system for functional biochemical characterization of candidate full-length P450 cDNAs. The system developed for the characterization of the PtAO enzyme involved a combination of yeast expression of P450 candidate cDNAs together with expression of cytochrome P450 reductase, in vivo feeding experiments with diterpene substrates, in vitro enzyme assays, and ultimately the development of an engineered yeast strain that produces conifer diterpene resin acids de novo based on the simultaneous expression of GGDP synthase, diterpene synthase, P450, and P450 reductase (Ro et al. 2005). Based on all current information, the biosynthesis of terpenoids in conifers involves several subcellular compartments (Keeling and Bohlmann 2006a). Enzymes of the MEV pathway are thought to be localized to the cytosol and endoplasmic reticulum, and the MEP pathway is localized in plastids. While GDP and GGDP synthases are

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most likely localized to plastids, FDP synthase is thought to reside in the cytosol and possibly in mitochondria. Based on the presence or absence of transit peptides, mono-TPS and di-TPS are also present in plastids, while sesqui-TPS appear to be cytosolic. Finally, the PtAO P450 is associated with the endoplasmic reticulum (Ro et al. 2005; Ro and Bohlmann 2006). It has been speculated that enzymes of terpenoid oleoresin formation are predominantly present in specialized epithelial cells lining the surface of resin ducts or resin blisters (Keeling and Bohlmann 2006b). However, testing this idea requires further validation by immuno-localization or cell-specific transcriptome and proteome analysis. Nevertheless, the involvement of several subcellular compartments in oleoresin terpenoid biosynthesis, the possible association of terpenoid biosynthesis with specialized cells, as well as the massive sequestration and accumulation of oleoresin terpenoids in the extracellular space of resin ducts or resin blister mandates efficient and specialized transport systems for intermediates and end-products of terpenoid biosynthesis. Such transport systems would be essential to deliver the large amounts of lipophilic terpenoids against steep concentration gradients and across cell membranes and the cell wall into the extracellular storage sites of specialized anatomical structures. The combined genomic and proteomic analysis of specialized cells types based on laser-assisted microdissection along with yeast expression systems developed for the de novo formation of conifer terpenoids in vivo (Ro et al. 2005) are now being explored for the discovery of candidate transport systems in conifer oleoresin biosynthesis and terpenoid accumulation.

8.3 Evolution of the Conifer TPS and P450 Gene Families for Terpenoid Defense The cloning and characterization of several dozen different TPS from several conifer species, including Norway spruce, Sitka spruce, loblolly pine, grand fir, and Douglas fir, enabled phylogenetic reconstruction of the gymnosperm TPS family, thereby shedding some light on the evolution of the larger TPS family in plants in general. Based on overall protein sequence relatedness, gene structure similarities, and catalytic mechanisms, all plant TPS are believed to have arisen from a common ancestor, which may have been closely related to the known conifer diTPS and to the recently characterized bifunctional ent-kaurene synthase from the moss Physcomitrella patens (Bohlmann et al. 1998a; Trapp et al. 2001b; Martin et al. 2004; Hayashi et al. 2006). It is possible that such an ancestral TPS was involved in the biosynthesis of the precursors of gibberellic acid, copalyl diphosphate and ent-kaurene. The evolution of the large family of plant TPS enzymes as we know them today occurred apparently to some large extend independently in the separate angiosperm and gymnosperm lineages involving numerous events of gene duplication and subsequent functional specialization, i.e. neo-functionalization and sub-functionalization (Martin et al. 2004; Keeling and Bohlmann 2006a; Keeling et al. 2008).


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Based on amino acid sequence similarity, the TPS family can be divided into seven subfamilies designated TPS-a through TPS-g (Bohlmann et al. 1998a; Martin et al. 2004). The conifer mono-TPS, sesqui-TPS and di-TPS cluster separately from the angiposperm TPS into the TPS-d subfamily, which is further divided into groups TPSd1 (mostly mono-TPS), TPS-d2 (mostly sesqui-TPS) and TPS-d3 (mostly di-TPS; all containing an ancestral 200-amino acid motif). Both phylogenetic analyses and analysis of gene structure position the conifer di-TPS closest to the putative ancestor of plant TPS (Bohlmann et al. 1998a; Trapp and Croteau 2001b; Martin et al. 2004). TPS of conifer secondary metabolism are believed to have evolved from TPS involved in primary metabolism. In the evolution of conifer TPS in terpenoid secondary metabolism a massive expansion of gene families and radiation of a diversity of biochemical functions has occurred (Martin et al. 2004). The same may be the case for the P450s (Hamberger and Bohlmann 2006). In contrast to the TPS and P450 of terpenoid secondary metabolism with their adaptive functions in conifer defense and resistance against insects and pathogens, to the best of current knowledge, the TPS and P450s in primary gibberellic acid phytohormone formation seem to be conserved and have undergone very little if any radiation in conifers.

8.4 Biosynthesis and Accumulation of Terpenoid Defenses Requires Specialized Cells Accumulation of large amounts of hydrophobic terpenoids in constitutive and induced (i.e., traumatic) oleoresin requires specialized anatomical structures in conifers stems, roots, foliage, and cones. Unless sequestered into extracellular spaces of such specialized structures, the hydrophobic terpenoids would interfere with biochemical processes, integrity of membranes and cell structure of the terpenoid producing cells. Sequestration and accumulation of terpenoid oleoresin can be achieved with simple, short-lived resin blisters or with complex, long-lived resin duct systems commonly found in conifer species that produce large quantities of oleoresin. As part of their ability to massively increase the biosynthesis and accumulation of terpenoid defenses, conifers also develop additional traumatic resin ducts (TRD) when challenged by insect attack or fungal inoculation or in response to simulated insect attack induced by treatment with MeJA or ethylene (e.g., Alfaro 1995; Nagy et al. 2000; Franceschi et al. 2002, 2005; Martin et al. 2002; Byun McKay et al. 2003; Hudgins et al. 2003; Krekling et al. 2004; Hudgins and Franceschi 2004; Huber et al. 2005b; Krokene et al. this volume). The de novo formation of TRD occurs within the cambium zone and outermost layers of developing xylem due to a transient change in cambial activity that initiates resin duct epithelial cells in lieu of wood-forming tracheids. Lumenal continuity between TRD, radial ducts, and resin ducts in the bark establishes a three-dimensional resin duct reticulum enabling enhanced biosynthesis, accumulation and flow of resin. The induced formation of TRD is associated with increased biosynthesis and accumulation of terpenoid oleoresin and involves induced gene expression and enzyme activities of several mono-TPS, sesqui-TPS, and di-TPS (Martin et al. 2002; Byun McKay et al. 2003, 2006; Fäldt

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et al. 2003; Huber et al. 2005b; Miller et al. 2005) and also involves increased levels of transcripts for octadecanoid and ethylene formation (Miller et al. 2005; Hudgins et al. 2006; Ralph et al. 2007a). Contact of TRD with ray parenchyma cells may enable signaling of induced terpenoid defenses between xylem, cambium, and bark tissues (Nagy et al. 2000; Franceschi et al. 2005; Hudgins et al. 2006; Ralph et al. 2007a). Given the abundance of relevant gene probes available from spruce EST and FLcDNAs projects (Ralph et al. 2006; Ralph and Bohlmann, in preparation) combined with immuno-localization, in-situ hybridization, and cell- or tissue specific micro-dissection techniques, it is now possible to test the cell-specific localization of transcripts and proteins for constitutive and induced terpenoid formation and its defense signaling in conifers (Hudgins et al. 2006; Keeling and Bohlmann 2006b; Ralph et al. 2007a).

8.5 Molecular Biology of Insect-and MeJA-Induced Terpenoid Defenses in Conifers The use of MeJA to induce chemical and anatomical defenses in conifers has been of critical importance in the characterization of insect-induced terpenoid defense responses in species of spruce (Franceschi et al. 2002; Martin et al. 2002, 2003a; Fäldt et al. 2003; Miller et al. 2005; Erbilgin et al. 2006; Zeneli et al. 2006). Similar to the effect of real insect attack or fungal inoculations, exogenous treatment of conifers with MeJA induces the development of TRD, terpenoid accumulation, as well as terpenoid volatile emissions. Treatment of trees with MeJA provided a means by which to induce conifer defense responses without mechanically injuring the tree. This is especially vital to the quantification of induced terpenoid defenses where any injury may reduce the biological capacity of the bark, cambium, or xylem tissue to respond, but would also enhance the loss of terpenoids through the wound site. Instead, treatment with MeJA allowed for the characterization of the induced enzyme activities and gene expression of terpenoid biosynthesis in stems and foliage of spruces, and enabled the detailed quantitative and qualitative analysis of terpenoids in control and induced trees. This approach also enabled the characterization of active emission of terpenoid volatiles from the needles of Sitka spruce and Norway spruce (Martin et al. 2002, 2003a; Miller et al. 2005). The cloning and functional characterization of families of TPS and P450 in species of spruce and other conifers provided an initial set of valuable probes for gene expression analysis of MeJA- or insect-induced terpenoid defenses (Fäldt et al. 2003; Byun McKay et al. 2003, 2006; Miller et al. 2005; Ro et al. 2005). TPS of all biochemical classes were induced by weevil-feeding or MeJA-treatment in stems of Sitka spruce, with some of the strongest responses observed for the mono-TPS and di-TPS. In loblolly pine, we also found a strong MeJA-induced increase of transcript levels of the PtAO P450 gene along with increased transcript levels of a diterpene synthase that produces the substrate precursor for PtAO (Ro et al. 2005; Ro and Bohlmann 2006). TPS gene expression and TPS enzyme activities are also elevated in foliage of Norway spruce and Sitka spruce following MeJA


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elicitation or weevil attack (Martin et al. 2003a; Miller et al. 2005). Induced gene expression of TPS in Norway spruce and Sitka spruce is associated with increased TPS enzyme activities and with the elevated accumulation of oleoresin terpenoids in stems or the release of terpenoid volatiles from needles (Martin et al. 2002; Miller et al. 2005). In recent work with Sitka spruce, we have established comprehensive maps of weevil-induced gene expression profiles for additional steps of terpenoid biosynthesis, indicating that TPS, P450, PTs, and a few genes in the MEP and MEV pathways are the strongest up-regulated transcripts in the weevil-induced terpenoid defense response in Sitka spruce (Ralph and Bohlmann, in preparation). In addition, microarray analyses on platforms with ∼21,800 spotted spruce cDNAs identified thousands of transcript species as differentially expressed in response to insect attack in Sitka spruce (Ralph et al. 2006; Ralph and Bohlmann, in preparation).

8.6 Effects of Conifer Terpenoids on Insects and Insect-Associated Pathogens Terpenoids in form of oleoresins and volatile emissions are a critical component of conifer interactions with other organisms such as defense and resistance against insects and pathogens (Langenheim 2003; Raffa et al. 2005). In general, terpenoid chemicals can protect conifers by providing mechanical barriers in form of ‘pitch’ or crystallized resin at wound sites, through toxicity against insects and insectassociated pathogens, or by interrupting essential processes in insect biology. In addition, in indirect defense systems, conifer terpenoids may function as signals for predators and parasites of the attacking herbivore. Specific functions of individual terpenoid compounds in conifer defense have been difficult to establish experimentally (Keeling and Bohlmann 2006a). While the complexity of hundreds of terpenoid chemicals that form the constitutive and inducible oleoresin blends is likely to provide a major advantage for the sustainability of a conifer defense system that is both stable and flexible, the same chemical complexity also poses a substantial challenge for researchers to pinpoint specific defense activities conclusively to individual terpenoid compounds. It is also possible that the mixture of terpenoids may be more effective than any individual compound alone. The lack of appropriate mutants for most conifers and the slow process of genetic transformation of conifers in general make it extremely difficult to manipulate complete terpenoid profiles or individual terpenoid compounds for in vivo elucidation of their defense functions with intact trees. Therefore, most tests of conifer terpenoids for their effects on insects or insect-associated pathogens have relied on exposure of insects or pathogens to isolated compounds or blends of these compounds. In such experiments, for example, the sesquiterpenoid compounds juvabione, farnesol, and farnesal have been shown to interrupt development and maturation of insects by interfering with insect endocrine systems (Schmialek 1963; Slama et al. 1965). The tree-killing activity of certain bark beetles involves an association of these insects with a community of fungi and bacteria some of which are known to be

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pathogenic and contribute directly to tree mortality. It is therefore important to establish the effect of terpenoid defenses with these pathogens as well. For example, the diterpene resin acids abietic acid and isopimaric acid strongly inhibit spore germination of the blue-stain fungus Ophiostoma ips, a conifer pathogen that is symbiotically associated with the pine engraver Ips pini (Kopper et al. 2005). The same study also showed that abietic acid inhibits mycelial growth of Ophiostoma ips. Several possible approaches can be considered for future research to test the role of individual terpenoids or groups of terpenoids in conifer defense. Targeted manipulation of terpenoid biosynthesis or genetic association studies are likely to provide the most informative approaches. Both approaches require funding commitments of many years, and genetic association studies also require access to substantial biological resources in form of established breeding programs and provenance trials. Fortunately, relevant biological materials are available to study the role of constitutive and traumatic terpenoids in the resistance of white spruce and Sitka spruce against the white pine weevil (Alfaro et al. 2002, 2004; King et al. 1997, 2004). In Sitka spruce, lines with extremely divergent profiles for individual terpenoids have been identified in metabolite profiling studies and the chemical profiles have been associated with resistance against the white pine weevil (Roberts, Keeling, and Bohlmann, unpublished results). In studies with white spruce, it has been established that insect-induced formation of traumatic resin ducts is positively correlated with resistance to white pine weevil (Alfaro et al. 1996, 2002; Tomlin et al. 1998). As far as targeted manipulation of terpenoids in conifers is concerned, as a proof of principle, we have recently transformed a sesquiterpenoid synthase, E-␣-bisabolene synthase, under the control of a wound- and insect-inducible promoter in white spruce (Godard et al. 2007). The use of an insect-inducible promoter will allow us to manipulate and test terpenoid defenses in a more realistic fashion than what can be accomplished with constitutive over-expression (Godard et al. 2007). Although terpenoids have important roles in the protection of conifers against insects, they are also vital for the successful attack against conifers by certain bark beetles and their associated tree-killing fungi. The large topic of conifer terpenoids and terpenoid pheromones as signals in bark beetle biology, as well as the topic of de novo formation of terpenoid pheromones in bark beetles have recently been addressed in several excellent reviews (e.g., Seybold and Tittiger 2003; Seybold et al. 2006; Raffa et al. 2005). It is well established that terpenoid volatiles emitted from conifer host trees function as semiochemicals in the identification of host and non-host conifer species as well as in the identification of individual susceptible trees within a given species. Once a tree has been attacked by a bark beetle, enzymes of the insect and the insect-associated microorganisms are required for detoxification of host terpenoid chemicals. A recent analysis of ESTs of the fungal pathogen Ophiostoma clavigerum, a blue-stain fungus associated with the mountain pine beetle (Dendroctonus ponderosa), revealed an overrepresentation of transcripts encoding for P450 and transport proteins in fungi exposed to terpenoids present in the conifer host tree (Diguistini et al. 2007). Beyond simple detoxification of terpenoid defenses, oxidation of monoterpenoids by bark beetles or by their associated fungi results in the formation of pheromones for the coordination


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of mass attack and mating of hundreds or thousands of insects on individual trees. Recent biochemical and molecular studies showed that bark beetle P450 enzymes play a central role in the conversion of conifer host defense chemicals into insect terpenoid pheromones (Huber et al. 2007; Sandstrom et al. 2006). A mass-attack by bark beetles that is coordinated by terpenoid pheromones allows the insect to overwhelm and exhaust the defense system of the much larger host tree and thus permits the insects and fungal pathogens to kill not only individual conifer trees but in epidemic situations leads to the destructions of conifer forest over landscape areas of millions of hectares. While much research has focused on the role of host monoterpenes as precursors for bark beetle sex and aggregation pheromones, it is now well established, based on precursor feeding experiments, enzyme characterization and gene discovery, that some species of coniferophagus bark beetles are able to produce monoterpenoid pheromones de novo (e.g., Gilg et al. 2005; Huber et al. 2007; Keeling et al. 2004, 2006; Martin et al. 2003b; Sandstrom et al. 2006; Seybold and Tittiger 2003; Seybold et al. 1995, 2000, 2006). In addition to the prominent role of terpenoids in the direct defense of conifers, indirect defense systems mediated by conifer volatiles have also been established in several conifers. For example, the egg deposition by the sawfly Diprion pini elicits emissions of terpenoid and other volatiles from Scots pine (P. sylvestris) which can result in the attraction of a parasitic wasp (Mumm and Hilker 2006). Over the millions of years of co-evolution of conifers with insects and insect-associated microorganisms, terpenoids may have played a substantial role in shaping the many interactions of conifers with insects and pathogens. Acknowledgments Research on conifer defenses in my laboratory has been generously supported with grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Human Frontiers Science Program (HFSP), Genome Canada, Genome British Columbia, and the Province of British Columbia. I wish to thank the members of my group, both past and present, for their many excellent contributions to our program and our collaborators in the BC Ministry of Forests and Range and the Canadian Forest Service for continuous support of the research in my group. Salary support has been provided, in part, by a UBC Distinguished University Scholar Award and an NSERC E.W.R. Steacie Memorial Fellowship. My research on conifer defense has been inspired by a very fine group of Washington State University eminent scholars: Dr. Rodney Croteau, who pioneered biochemical research on conifer terpenoids; Dr. Vince Franceschi, who introduced me to the fascinating system of traumatic resin cells; and last but not least Dr. Clarence (Bud) Ryan whose pioneering work on induced plant defense against insects provided a foundation for my research on induced defenses in conifers.

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Hamberger B, Bohlmann J (2006) Cytochrome P450 monooxygenases in conifer genomes: Discovery of members of the terpenoid oxygenase superfamily in spruce and pine. Biochem Soc Transactions 34:1209–1214 Hayashi KI, Kawaide H, Notomi M, Sagiki Y, Matsuo A, Nozaki H (2006) Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens. FEBS Lett 580:6175–6181 Huber DPW, Philippe RN, Godard KA, Bohlmann J (2005a) Characterization of four terpene synthase cDNAs from methyl jasmonate-induced Douglas-fir, Pseudotsuga menziesii. Phytochemistry 66:1427–1439 Huber DPW, Phillippe RN, Madilao L, Sturrock RN, Bohlmann J (2005b) Changes in anatomy and terpene chemistry in roots of Douglas-fir seedlings following treatment with methyl jasmonate. Tree Physiol 25:1075–1083 Huber DPW, Erickson ML, Leutenegger CM, Bohlmann J, Seybold SJ (2007) Isolation and extreme sex-specific expression of cytochrome P450 genes in the bark beetle, Ips paraconfusus, following feeding on the phloem of host ponderosa pine, Pinus ponderosa. Insect Mol Biol 16:335–349 Hudgins JW, Christiansen E, Franceschi VR (2003) Methyl jasmonate induces changes mimicking anatomical defenses in diverse members of the Pinaceae. Tree Physiol 23:361–371 Hudgins JW, Franceschi VR (2004) Methyl jasmonate-induced ethylene production is responsible for conifer phloem defense responses and reprogramming of stem cambial zone for traumatic resin duct formation. Plant Physiol 135:2134–2149 Hudgins JW, Ralph SG, Franceschi VR, Bohlmann J (2006) Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells. Planta 224:865–877 Keeling CI, Blomquist GJ, Tittiger C (2004) Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae). Naturwissenschaften 91:324–328 Keeling CI, Bohlmann J (2006a) Genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defence of conifers against insects and pathogens. New Phytol 170:657–675 Keeling CI, Bohlmann J (2006b) Diterpene resin acids in conifers. Phytochemistry 67:2415–2423 Keeling CI, Bearfield JC, Young S, Blomquist GJ, Tittiger C (2006) Effects of juvenile hormone on gene expression in the pheromone-producing midgut of the pine engraver beetle, Ips pini. Insect Mol Biol 15:207–216 Keeling CI, Weißhaar S, Lin R, Bohlmann J (2008) Functional plasticity of paralogous diterpene synthases involved in conifer defense. Proc Natl Acad Sci USA, in press King JN, Yanchuk AD, Kiss GK, Alfaro RI (1997) Genetic and phenotypic relationships between weevil (Pissodes strobi) resistance and height growth in spruce populations of British Columbia. Can J For Res 27:732–739 King JN, Alfaro RI, Cartwright C (2004) Genetic resistance of Sitka spruce (Picea sitchensis) populations to the white pine weevil (Pissodes strobi): distribution of resistance. Forestry 4:269–278 Kopper BJ, Illman BL, Kersten PJ, Klepzig KD, Raffa KF (2005) Effects of diterpene acids on components of a conifer bark beetle-fungal interaction: tolerance by Ips pini and sensitivity by its associate Ophiostoma ips. Environ Entomol 34:486–493 Krekling T, Franceschi VR, Krokene P, Solheim H (2004) Differential anatomical response of Norway spruce stem tissues to sterile and fungus infected inoculations. Trees-Struct Funct 18:1–9 Langenheim JH (2003) Plant resins: chemistry, evolution, ecology, and ethnobotany. Timber Press Inc, Portland, OR, p 586 Lippert D, Chowrira S, Ralph SG, Zhuang J, Aeschliman D, Ritland C, Ritland K, Bohlmann J (2007) Conifer defense against insects: proteome analysis of Sitka spruce (Picea sitchensis) bark induced by mechanical wounding or feeding by white pine weevils (Pissodes strobi). Proteomics 7:248–270

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Martin D, Tholl D, Gershenzon J, Bohlmann J (2002) Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of Norway spruce stems. Plant Physiol 129:1003–1018 Martin DM, Gershenzon J, Bohlmann J (2003a) Induction of volatile terpene biosynthesis and diurnal emission by methyl jasmonate in foliage of Norway spruce (Picea abies). Plant Physiol 132:1586–1599 Martin D, Bohlmann J, Gershenzon J, Francke W, Seybold SJ (2003b) A novel sex-specific and inducible monoterpene synthase activity associated with a pine bark beetle, the pine engraver, Ips pini. Naturwissenschaften 90:173–179 Martin DM, Fäldt J, Bohlmann J (2004) Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol 135:1908–1927 Martin D, Bohlmann J (2005) Molecular biochemistry and genomics of terpenoid defenses in conifers. Rec Adv Phytochem 39:29–56 Miller B, Madilao LL, Ralph S, Bohlmann J (2005) Insect-induced conifer defense. White pine weevil and methyl jasmonate induce traumatic resinosis, de novo formed volatile emissions, and accumulation of terpenoid synthase and octadecanoid pathway transcripts in Sitka spruce. Plant Physiol 137:369–382 Mumm R, Hilker M (2006) Direct and indirect chemical defense of pine against folivorous insects. Trends Plant Sci 11:351–358 Nagy NE, Franceschi VR, Solheim H, Krekling T, Christiansen E (2000) Wound-induced traumatic resin duct development in stems of Norway spruce (Pinaceae): anatomy and cytochemical traits. Am J Bot 87:302–313 Phillips MA, Croteau RB (1999) Resin-based defenses in conifers. Trends Plant Sci 4:184–190 Phillips MA, Wildung MR, Williams DC, Hyatt DC, Croteau R (2003) cDNA isolation, functional expression, and characterization of (+)-␣-pinene synthase and (–)-␣-pinene synthase from loblolly pine (Pinus taeda): Stereocontrol in pinene biosynthesis. Arch Biochem Biophys 411:267–276 Phillips MA, Bohlmann J, Gershenzon J (2006) Molecular regulation of induced terpenoid biosynthesis in conifers. Phytochem Rev 5:179–189 Phillips MA, Walter MH, Ralph SG, Dabrowska P, Luck K, Urós EM, Boland W, Strack D, Rodr´ıguez-Conceptión M, Bohlmann J, Gershenzon J (2007) Functional identification and differentential expression of 1-deoxy-D-xylulose-5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies). Plant Mol Biol 65:243–257 (doi:10.1007/s11103007-9212-5) Raffa KF, Aukema BH, Erbilgin N, Klepzig KD, Wallin KF (2005) Interactions among conifer terpenoids and bark beetles across multiple levels of scale: An attempt to understand links between population patterns and physiological processes. Rec Adv Phytochem 32: 79–118 Ralph SG, Yueh H, Friedmann M, Aeschliman D, Zeznik JA, Nelson CC, Butterfield YSN, Kirkpatrick R, Liu J, Jones SJM, Marra MA, Douglas CJ, Ritland K, Bohlmann J (2006) Conifer defense against insects: Microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome. Plant Cell Environ 29:1545–1570 Ralph SG, Hudgins JW, Jancsik S, Franceschi VR, Bohlmann J (2007a) Aminocyclopropane carboxylic acid synthase is a regulated step in ethylene-dependent induced conifer defense. Full-length cDNA cloning of a multigene family, differential constitutive, and wound- and insect-induced expression, and cellular and subcellular localization in spruce and Douglas fir. Plant Physiol 143:410–424 Ralph SG, Jancsik S, Bohlmann J (2007b) Dirigent proteins in conifer defense II: Extended gene discovery, phylogeny, and constitutive and stress-induced gene expression in spruce (Picea spp.). Phytochemistry 68:1974–1990


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Chapter 9

Phenylpropanoid Metabolism Induced by Wounding and Insect Herbivory Mark A. Bernards and Lars B˚astrup-Spohr

Wounding of plant tissue, whether by biotic or abiotic means, results in a massive re-arrangement of metabolism, with the primary objectives of isolating the affected tissue and limiting the extent of further damage. Within this context, newly synthesized phenylpropanoid-derived compounds are known to provide anti-feedant, anti-microbial, and cytotoxic effects, as well as building blocks for structural macromolecules (e.g., lignin, suberin) and condensed tannins. The literature describing hydroxycinnamates (and their conjugates), flavonoids and condensed tannins vis a vis their roles in plant/herbivore interactions is incomplete. Nevertheless, it is clear that the biosynthesis of phenylalanine, a necessary precursor, is induced by herbivore damage, and there is a coordinate induction of transcripts/enzymes associated with hydroxycinnamic acid, flavonoid, and condensed tannin formation. Recent genome scale studies have provided evidence for the involvement of many genes in the response to herbivory, including those of phenylpropanoid metabolism. The next steps will certainly involve the functional characterization of these genes. Forward progress is dependent on expanding analyses to more plant-insect interactions, as well as monitoring plant reactions to herbivory (or wounding) over relevant time frames. Defining the biochemistry of the phenylpropanoid metabolism of specific plant-insect interactions, both at the transcript and enzyme levels, will lead to the discovery of new pathways, or variations on known pathways. In defining plant/herbivore-specific variations in phenylpropanoid metabolism we will discover the underlying strategies used by plants to defend themselves and gain insights into the role(s) of phenylpropanoids in these processes.

9.1 Introduction The overall success of plants defending themselves against herbivores likely depends on a combination of both pre-formed defenses (phytochemicals, physical barriers, etc.) and those induced by the chewing, boring, or sucking of insects. M.A. Bernards Department of Biology, The University of Western Ontario, London, ON, Canada, N6A 5B7 e-mail: [email protected] A. Schaller (ed.), Induced Plant Resistance to Herbivory, C Springer Science+Business Media B.V. 2008

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This chapter summarizes the field of wound- and insect herbivore-induced phenylpropanoid metabolism, with an emphasis on the types of molecules involved, and the enzymes and pathways associated with their biosynthesis. The chapter begins with an overview of the types of phenolic compounds known to accumulate in response to wounding or herbivory, and some of their putative roles in defense against herbivorous insects, followed by a summary of the literature describing the enzymology of phenylpropanoid metabolism induced by wounding and/or herbivory. It is important to note up front that the literature on herbivore-induced phenylpropanoid enzymes at the protein/enzyme activity level is sparse. Few contributions describing the enzymology of wound- and/or herbivory-induced phenylpropanoid metabolism have been made since the summary chapter written by Constable (1999). Instead, much of the recent literature has focused on the induction of specific genes at the transcript level and the use of genome wide screening of herbivory and wound-induced genes using available microarrays. With the latter, there is much reliance on database annotation for the identification of genes, with only a small subset of these having been verified by functional analysis, so far. As we look ahead to what needs to be accomplished in the future to further our understanding of the role of phenolics in the overall interaction between plants and their insect pests, functional characterization of the dozens of genes putatively involved in induced phenylpropanoid metabolism will be high priority. Since there are chapters elsewhere in this book that specifically address volatile aromatics (Chapters 19–21), wound periderm (Chapter 6), and polyphenol oxidase (Chapter 12), these topics will not be repeated herein. Similarly, there is an entire section in this book devoted to signals in defense; as such the topic of woundinduced signaling (e.g., via the jasmonate pathway) will not be covered.

9.2 Phenolic Compounds and Plant-Herbivore Interactions 9.2.1 Roles for Phenolics in Plant-Herbivore Interactions Phenolic compounds are aromatic compounds bearing one or more hydroxyl group on an aromatic ring. Phenylpropanoids, strictly speaking, are phenolic compounds derived from phenylalanine. And while there are >8000 known phenolic compounds, roughly categorized into 14 compound classes based on the number of carbons and their arrangement (e.g., Strack 1997), only a small fraction of them have been implicated in plant-herbivore interactions. Amongst these are the benzoic acids, hydroxycinnamic acids (and their conjugates), furanocoumarins, coumarins, stilbenes, flavonoids (especially flavonols), hydrolysable tannins, condensed tannins (catechin polymers; Fig. 9.1), and lignin (reviewed in Constable 1999). Wounding and herbivory also induce the formation of other poly(phenolic)-based cell wall modifications, especially suberin (Ginzberg this volume).

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Fig. 9.1 Representative phenolics associated with wounding and herbivory. Examples from the main classes of phenolic compounds known to be associated with either the response of plants to mechanical wounding and/or herbivory are shown. For the tannins, repeating phenolic units are shown in square brackets. See text for details

Different phenolic compounds play different roles in the plant-herbivore interaction. For example, hydroxycinnamic acids may act as cell wall cross-links that fortify and protect plant cell walls against chewing damage (Santiago et al. 2005, 2006). Tannins, which accumulate in many plant species, in response to herbivory, especially within trees where they are most common (Bernays et al. 1989; Peters and Constabel 2002), exert their biological effect through protein binding. They have been shown to affect a wide variety of herbivores from insects to mammals (for a review see Bernays et al. 1989). In insects, tannins can work by producing lesions in the midgut of these animals. Insects that normally feed on tannins have been found to have a relatively thick protective peritrophic membrane lining the midgut epithelium. This is thought to be evolutionary evidence that tannins play a role in plant-insect interactions. Tannins are mainly found in woody species and therefore primarily exert their effect on species that forage on trees (Bernays et al. 1989; Peters and Constabel 2002). Condensed and non-condensed tannins contribute to the overall oxidative potential of cells (e.g., Barbehenn et al. 2005, 2006), as do other flavonoids (reviewed

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in Treutter 2005). Paradoxically, one of the hallmark features of phenylpropanoids in general is their anti-oxidative properties (i.e., they are readily oxidized to relatively stable quinone intermediates), but in the context of resistance to herbivores this translates into a class of compounds that, once oxidized can interfere with proteins in the digestive tract of chewing insects and mammals. This can result in an overall decline in nutritional quality of plant material and/or cause oxidative stress in herbivores (Bi and Felton 1997; Barbehenn et al. 2005, 2006). The effect of defense compounds on herbivores also varies according to the compound. For example, defense compounds can influence herbivore settling, feeding, oviposition, growth fecundity and/or fertility (Walling 2000). Since several types of phenolic compounds (e.g., hydroxycinnamates, hydroxycinnamoyl conjugates, flavonoids) possess anti-microbial properties (e.g., Malmberg 1984; Ding et al. 2000), their accumulation in tissue damaged by wounding (including chewing damage) could play a dual role: direct defense against herbivores and protection against opportunistic pathogens. For example, in the wound response of potato tubers, where the predominant role of induced phenylpropanoid metabolism appears to be associated with the formation of the poly (phenolic) domain of suberin, other phenolics (e.g., hydroxycinnamoyl amides, chlorogenic acid) that are not only involved in suberin formation also accumulate (Cottle and Kolattukudy 1982; Malmberg 1984; Razem and Bernards 2002). The suberin phenolics play a role in forming a physical barrier, while the soluble phenolics possess anti-microbial properties and presumably protect against opportunistic pathogens.

9.2.2 Constitutive vs. Induced Accumulation of Phenolics Phenolics are widely distributed in plants, and accumulate during normal growth and development. Consequently, phenolics are constitutively present in plants prior to insect or mammalian herbivory-induced damage (i.e., wounding). Indeed, roles for phenolics as pre-formed (constitutive) defenses against herbivory are well documented (e.g., Ding et al. 2000; Mutikainen et al. 2000; Treutter 2005; Santiago et al. 2005, 2006) and include physical barriers such as cell wall bound phenolics, lignins, suberin, and cuticle-associated phenolics as well as stored compounds that have a deterring (anti-feedant) or directly toxic (insecticidal) effect on herbivores (Walling 2000). Induced defenses, on the other hand, are characterized by the synthesis of new compounds or structural barriers after herbivore-induced damage has occurred, and includes both proteins and secondary metabolites. Induced defenses can work both locally, near the point of attack and/or systemically, i.e., throughout the plant. Since producing defense related compounds is metabolically expensive, constitutive and induced defense mechanisms can be seen as two different strategies for avoiding herbivores. Constitutive defenses are always present, but potentially energetically costly. For example, tannins accumulate as constitutive defense in poplar leaves (Arnold et al. 2004), but the extent of accumulation is dependent on the sink


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strength of the developing leaves and associated with the import of carbohydrates from older source leaves. If other sinks, such as the roots, are removed by girdling and thus decreasing competition for the carbohydrates, an increase in leaf tannin concentration occurs. Increased phenolic accumulation in association with high sink strength, does not appear to be universal, however, since the opposite phenomenon has been found in the gymnosperm species Pinus sylvestris, where the production of phenolics was negatively correlated with sink strength (Honkanen et al. 1999). Similarly, phenolics can also be induced in poplar leaves as a response to grazing by gypsy moth larvae, mechanical wounding and treatment with jasmonic acids (Arnold et al. 2004). In this study phenolic responses in leaves, induced by herbivores, were found in the first six to eight days post emergence, when the whole canopy was treated with jasmonic acid. During this period leaves are characterized by sink strength indicated by the activity of the enzyme cell wall invertase that facilitates phloem unloading to the cells. Arnold et al. (2004) found that when carbohydrate source leaves were removed, the induced response to jasmonic acid was significantly reduced. These results indicate that even a local defense response in developing foliage depends to some degree on long distance carbohydrate transport from source tissue. Thus, the role of tannins in plant defense, and maybe other phenylpropanoids as well, is not only dependent on the plant’s genetic basis to produce them, but also on what stage a particular leaf is in and if there is a source of carbohydrates to supply the production. Induced defenses are only invoked after tissue damage has occurred. While this potentially provides defense at a lower energetic cost it also comes with some loss of biomass. Thus plants need to balance the allocation of carbon and nitrogen resources between these two types of defenses and vegetative and reproductive growth to insure survival in the long term (Walling 2000). However, it is not always easy to distinguish between constitutive and induced defense mechanisms since some defenserelated compounds are constitutively synthesized and stored, and also synthesized de novo as a response to herbivore damage (Ding et al. 2000; Gatehouse 2002). To add to this complexity, induced metabolic pathways differ when plants are exposed to different types of herbivory, and even species of herbivores. Generally insects that use a piercing/sucking mode of feeding elicit a response similar to that of fungal and bacterial plant pathogens, while chewing insects produce a response similar to that of mechanical wounding (e.g., Walling 2000). In the case of chewing insects, it has been reported that the damage made by insects is not equivalent to mechanical wounding alone (Hartley and Firn 1989) since, for example, insect regurgitant can increase the response to chewing herbivory over that induced by mechanical wounding alone (Korth and Dixon 1997). Further to this, some plants react specifically to one species of herbivore or even a specific life stage within a species (van der Ven et al. 2000), which may correspond to the content of the saliva in the insect species (Walling 2000). However, the general response of plants to wounding and herbivore damage, vis a vis induced phenylpropanoid metabolism and the accumulation of phenolic compounds, is essentially the same: i.e., the same types of compounds are induced to accumulate, and thereby the same enzymes involved. Notwithstanding the subtle details defining specific plant-herbivore interactions, the

194


wound response literature, which is more complete than that of herbivore response literature, can provide a model for the phenylpropanoid metabolic processes involved in the response of plants to herbivore damage.

9.3 Induced Biosynthesis of Phenolic Compounds Historically, phenylpropanoid metabolism has been divided into two main stages, ‘general phenylpropanoid metabolism’ (i.e., the biosynthesis of hydroxycinnamic acids) and pathway specific metabolism (i.e., hydroxycinnamic acids as precursors to specific classes of phenylpropanoids such as flavonoids or coumarins; e.g., Strack 1997). More recently it has been shown that the interconversion between hydroxycinnamic acids does not necessarily lie within a defined general pathway (reviewed in Dixon et al. 2001; Humphreys and Chapple 2002), and in fact branches into compound class-specific pathways as early as cinnamate (or cinnamoyl-CoA), or after its hydroxylation to form p-coumarate. Consequently, ‘general phenylpropanoid metabolism’ only consists of two common steps: the deamination of phenylalanine by phenylalanine ammonia-lyase (PAL) and the 4-hydroxylation of cinnamate by cinnamate-4-hydroxylase (C4H). The main classes of soluble phenolic compounds involved in the response of plants to wounding and herbivory are hydroxycinnamic acids (and their conjugates and derivatives) and tannins (via flavonoids), and these will be considered separately below. However, phenylpropanoids are derived from phenylalanine, which also has to be synthesized de novo as part of the overall wound/herbivory response, and it is important to consider this as an early stage of induced phenylpropanoid metabolism.

9.3.1 The Shikimate Pathway All three aromatic amino acids, phenylalanine, tyrosine, and tryptophan are synthesized via the shikimate pathway. With respect to induced phenolic metabolism, the synthesis of phenylalanine (Fig. 9.2; Table 9.1) is of particular importance since a significant flux of carbon must go through this intermediate. The first enzyme in the shikimate pathway is 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, which catalyses the condensation of one molecule each of phosphoenolpyruvate and erythrose-4-phosphate; both of these starting materials are derived from primary metabolism. DAHP synthase has been shown to be induced, at the transcript, protein, and enzyme activity levels, in wounded potato tubers and tomato fruits (Dyer et al. 1989). More recently, DAHP synthase gene up-regulation in Sitka spruce bark following mechanical wounding and weevil feeding, as well as in buds following spruce bud worm larvae feeding has been demonstrated at the transcript level, using a microarray gene profiling approach (Ralph et al. 2006). In addition, shikimate kinase was also shown to be coordinately up-regulated with DAHP synthase in the same tissues (Ralph et al. 2006). Similarly, wounding has been


195

Fig. 9.2 The shikimate pathway and N-recycling in plants. The shikimic acid pathway, from phospho-enol-pyruvate and erythrose-4-phosphate to phenylalanine is shown. Cofactors and cosubstrates are not shown for clarity. Refer to Table 9.1. Bold arrows indicate steps for which wounding and/or herbivory have been shown to result in induced gene expression and/or enzyme activity. Enzymes are: (1) 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase; (2) 3-dehydroquinate synthase; (3) 3-dehydroquinate dehydratrase; (4) shikimate dehydrogenase; (5) shikimate kinase; (6) 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase;

196


shown to induce enzyme activity levels of dehydroquinate dehydratase/shikimate dehydrogenase in sweet potato (Minamikawa et al. 1966) and chorismate mutase in potato tubers (Kuroki and Conn 1988). These studies provide a critical link between primary carbon metabolism and the de novo synthesis of phenolics in response to wounding and herbivory. No other enzymes of the shikimate pathway have been shown to be directly involved in the response of plants to wounding or herbivory, either at the transcript or protein levels. However, a recent genomic-scale exploration of gene expression during active lignin formation in poplar (Ehlting et al. 2005) has demonstrated that several shikimate pathway genes are co-coordinately regulated, including 3-dehydroquinate synthase, 3-dehydroquinate dehydratrase/shikimate dehydrogenase, shikimate kinase, chorismate synthase, chorismate mutase, putative aspartate: prephenate aminotransferases and arogenate dehydratase. Significantly, neither arogenate dehydrogenase (which forms tyrosine from arogenate), nor any of the genes associated with tryptophan biosynthesis (which branches off of the main shikimate pathway at chorismate) were up-regulated during lignification, suggesting a process-specific subset of shikimate pathway genes to be involved. The synchronized upregulation of genes for shikimate pathway enzymes and PAL was also observed in tomato cell cultures in response to fungal elicitors (Görlach et al. 1995). Thus, while not strictly wound- or herbivory-based studies, these data illustrate the coordinate regulation of the biosynthetic pathway providing the precursor for phenylpropanoid metabolism. Presumably, this level of coordinate regulation is in operation when phenylpropanoid metabolism is induced by wounding or herbivore damage. Herbivory and wounding have been clearly linked to the induction of PAL at the transcript, protein and enzyme levels (e.g., Bi and Felton 1995; Hartley and Firn 1989; Bernards et al. 2000; Devoto et al. 2005; Qiuju et al. 2005; Saltveit et al. 2005; Ralph et al. 2006). Phenylalanine ammonia-lyase serves as the gateway enzyme into phenylpropanoid metabolism, and while it is considered in more detail in Section 9.3.2, it is mentioned here since metabolism of phenylalanine represents an exit point from the shikimate pathway and provides a strong metabolic draw on it. The deamination of phenylalanine by PAL yields two products: cinnamic acid, the metabolic fate of which will be discussed in greater detail in Section 9.3.2, and ammonia. As noted above, the production of defense compounds is metabolically expensive, and the ‘cost’ of inducing phenylpropanoid metabolism for chemical defense could potentially be very high if one mole of ammonia were lost for each mole of phenylpropanoid metabolite formed. However, plants have evolved an efficient N-recycling mechanism in which the ammonia liberated from phenylalanine by PAL is reused in the production of more phenylalanine (Fig. 9.2). This recycling

Fig. 9.2 (continued) (7) chorismate synthase; (8) chorismate mutase; (9) aspartate:prephenate aminotransferase; (10) arogenate dehydratase; (11) phenylalanine ammonia-lyase; (12) glutamine synthetase; (13) ferredoxin-dependent glutamine:2-oxoglutarate aminotransferase. ␣-KG, ␣-ketoglutarate (2-oxoglutarate); Fdxred , reduced ferredoxin; Fdxox , oxidized ferredoxin. (Adapted in part from Razal et al. 1996; Strack 1997; Croteau et al. 2000)

4.2.3.5

1.4.7.1

6.3.1.2

4.3.1.5

glutamate + NH+ 4 + ATP → glutamine + ADP + Pi glutamine + 2-oxoglutarate + Fdxred → 2 glutamate + Fdxox

phenylalanine → cinnamate + NH+ 4

5-enolpyruvylshikimate-3-phosphate → chorismate + Pi chorismate → prephenate prephenate + aspartate → arogenate + oxaloacetate arogenate → phenylalanine + CO2 + H2 O

shikimate-3-phosphate + phosphoenolpyruvate → 5-enolpyruvylshikimate-3-phosphate + Pi

At1g22410, At4g33510, At4g39980

phosphoenolpyruvate + erythrose 4-phosphate + H2 O → 3-Deoxy-D-arabinoheptulosonate-7-phosphate + Pi 3-Deoxy-D-arabino-heptulosonate-7-phosphate → 3-dehydroquinate + Pi 3-dehydroquinate → 3-dehydroshikimate + H2 O 3-dehydroshikimate + NADP+ → shikimate + NADPH,H+ shikimate + ATP → shikimate-3phosphate + ADP

At1g48470, At1g66200, At3g17820, At3g53180, At5g16570, At5g35630, At5g37600 At2g41220

At1g08250, At1g11790, At2g27820, At3g07630, At3g44720, At5g22630 At2g37040, At3g10340, At3g53260, At5g04230

At1g69370, At3g29200, At5g10870 Unknown in Arabidopsis

At1g48850

At1g06890, At1g60530, At2g14050, At2g16790, At2g21940, At2g35500, At3g50950, At3g26900, At4g39540, At5g22370, At5g45210, At5g47050 At1g48860, At2g45300

At3g06350

At5g66120

Representative Arabidopsis genes2

Reaction catalyzed

2

Enzyme Nomenclature (web version) www.chem.qmul.ac.uk/iubmb/enzyme/ Gene identifiers are from Ehlting et al. 2005, Ralph et al. 2006, and The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org). Not all genes listed have been functionally characterized; nor are the lists considered complete. 3 Enzyme number refer to the corresponding reaction in Fig. 9.2.

1

13 Ferredoxin-dependent glutamate synthase

11 Phenylalanine Ammonia-lyase (PAL) 12 Glutamine Synthetase (GS)

Chorismate Mutase (CM) 5.4.99.5 Aspartate-Prephenate 2.6.1.78 Aminotransferase 10 Arogenate Dehydratase (ADT) 4.2.1.91

8 9

7

5-enolpyruvylshikimate 3-phosphate Synthase (EPSPS) Chorismate Synthase (CS)

6

2.5.1.19

2.7.1.71

Shikimate Kinase (SK)

5

4.2.3.4

2.5.1.54

4.2.1.10/ 1.1.1.25

3-Deoxy-D-arabinoheptulosonate-7-phosphate Synthase (DAHPS) 3-Dehydroquinate Synthase

E.C number1

3/4 3-Dehydroquinate Dehydratase/ Shikimate Dehydrogenase

2

1

3

Enzyme name

Table 9.1 Enzymes of the shikimate pathways leading to phenylalanine and their corresponding genes in Arabidopsis 9 Wound-Induced Phenylpropanoid Metabolism 197

198


mechanism, which involves the glutamine synthetase/ferredoxin-dependent glutamine 2-oxoglutarate amino transferase (GS/GOGAT) ammonia assimilation system, has been demonstrated in wound healing potato tubers (Razal et al. 1996), sweet potato tubers (Singh et al. 1998), and in lignifying cell cultures of Pinus taeda (van Heerden et al. 1996). Thus, the heavy demand for phenylalanine brought about by wound-induced phenylpropanoid metabolism is met by the induction of the shikimate pathway and an efficient N-recycling system. However, with the exception of demonstrated GS up-regulation post wounding and/or herbivore damage in Sitka spruce (Ralph et al. 2006), these aspects of induced phenylpropanoid metabolism have not been demonstrated in plant-herbivore interactions. While it is logical to assume that the shikimate and N-recycling pathways are more broadly associated in plant-herbivore interactions resulting in de novo synthesis of phenolics, confirmation of their participation is lacking.

9.3.2 Induced General Phenylpropanoid Metabolism and the Origin of Hydroxycinnamates The induction of phenylpropanoid metabolism is implied in the accumulation of newly formed phenolic compounds in several plants in response to herbivore damage, e.g., p-coumaric and ferulic acids in wheat exposed to Sitodipolis mosellana (Ding et al. 2000) and corn exposed to Sesamina noonagrioides (Santiago et al. 2005, 2006), tannins in many tree species (Bernays et al. 1989; Peters and Constabel 2002; Arnold et al. 2004), and phenolics in Medicago truncatula damaged by Spodoptera littoralis (Leitner et al. 2005). There are relatively few reports in which the activity of phenylpropanoid biosynthesis enzymes is measured directly. Phenylalanine ammonia-lyase (PAL) is the gateway enzyme providing C6 –C3 carbon skeletons for the formation of essentially all phenylpropanoids and their derivatives (Fig. 9.3, Table 9.2). The conversion of Phe to cinnamate + NH+ 4 is considered to be physiologically irreversible, and thus is a good indicator of carbon commitment to phenylpropanoid metabolism. Phenylalanine ammonia-lyase is represented by a small gene family (reviewed in Dixon and Paiva 1995; Dixon et al. 2001). For example, at least four genes have been annotated as PAL in the Arabidopsis database, while ten uniquely expressed sequence tags (ESTs) are described for Sitka spruce (Ralph et al. 2006). Not surprisingly, this step in the phenylpropanoid metabolic pathway has received a great deal of attention in the literature, albeit largely as a function of biotic and abiotic stress induction; e.g., response to pathogen attack, exposure to UV light, wounding etc. (Strack 1997; Croteau et al. 2000). From a herbivory induction standpoint, increased PAL activity has been directly measured in a few insect/plant systems, e.g., Helicoverpa zea damaged soybean leaves (Bi and Felton 1995); Apocheima pilosaria damaged birch (Hartley and Firn 1989); gypsy moth larvae damaged poplar leaves (Arnold et al. 2004). At the transcript level, PAL has been shown to be induced by insect damage of Arabidopsis (Reymond et al. 2004) and Sitka spruce (Ralph et al. 2006). There are many more

199

Fig. 9.3 (continued)


200


Fig. 9.3 (continued) Biosynthesis of hydroxycinnamic acids and their conjugates and derivatives. The biosynthesis of hydroxycinnamates, hydroxycinnamoyl conjugates, and derivatives of hydroxycinnamates is depicted. Cofactors and co-substrates are not shown for clarity. Refer to Table 9.2. Bold arrows indicate steps for which wounding and/or herbivory have been shown to result in induced gene expression and/or enzyme activity, while dashed arrows are hypothetical steps. Enzymes are: (11) phenylalanine ammonia-lyase; (14) cinnamate-4-hydroxylase; (15) 4-coumaroylCoA ligase; (16/16a) coumarate-3-hydroxylase; (17) caffeoyl-CoA O-methyltransferase; (18/18a) ferulate-5-hydroxylase; (19/19a) caffeic acid/5-hydroxyferulic acid O-methyltransferase; (20) hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyltransferase; (21) cinnamoyl-CoA reductase; (22) cinnamyl alcohol dehydrogenase; (23) hydroxycinnamoyl CoA:tyramine hydroxycinnamoyltransferase; (24) p-coumaroyltyramine-3-hydroxylase; (25) caffeoyltyramine O-methyltransferase; (26) caffeoyl CoA:quinate caffeoyltransferase. (Adapted in part from Strack 1997; Croteau et al. 2000; Humphreys and Chapple 2002)

examples of wound-induced PAL, both at the transcript expression level (e.g., Reymond et al. 2000; Devoto et al. 2005; Ralph et al. 2006) and enzyme level (e.g., Bernards et al. 2000; Qiuju et al. 2005; Saltveit et al. 2005). Cinnamate-4-hydroxylase (C4H) catalyses the first of potentially many hydroxylation reactions in phenylpropanoid metabolism, generating the first ‘phenolic’ compound, p-coumaric acid, in the phenylpropanoid metabolic pathway (Fig. 9.3). It is a cytochrome P-450 monooxygenase of the CYP73A subfamily. Cinnamate4-hydroxylase is induced at the transcript level in wound- and insect-damaged Sitka spruce (Ralph et al. 2006) and lignifying poplar stems (Ehlting et al. 2005). Similarly, transcription of C4H in jerusalem artichoke (Batard et al. 1997) and Camptotheca acuminata leaves (Kim et al. 2005) in response to wounding, has been demonstrated. The formation of p-coumaric acid represents a critical step in phenylpropanoid metabolism since this intermediate can be utilized as the precursor to a myriad of end products (Figs. 9.3 and 9.4). For example, p-coumaroyl-CoA is the direct precursor to all flavonoids (Fig. 9.4), and ultimately the condensed tannins (Section 9.3.3) as well as the monolignols (via p-coumaroylquinnate or pcoumaroylshikimate intermediates) and potentially, other hydroxycinnamoyl conjugates (e.g., hydroxycinnamoyltyramines; Fig. 9.3). It is also the common substrate for further hydroxylation and methylation to generate characteristic aromatic substitution patterns of phenolics. Historically, hydroxylation of the p-coumaroyl sub-structure has been depicted as occurring at the level of the coenzyme-A thioester; however, in the context of monolignol formation, it has recently been shown that this reaction proceeds after ester transfer to shikimate or quinate (Schoch et al. 2001). Regardless, coenzyme-A thioester formation, via 4-coumaroyl-CoA ligase (4CL), is an important activation step. As with PAL, 4CL is represented by a gene family in plants, with a range of 4CL-like genes identified via sequence similarity analysis, and observation of their coordinate regulation during lignification (Ehlting et al. 2005). Amongst these, at least fifteen 4CL/4CL-like genes are induced by wounding, weevil damage and/or budworm damage in Sitka spruce (Ralph et al. 2006). Similarly, 4CLs are induced by wounding at the transcript level in several other plant species, including parsley and tobacco carrying one of the parsley genes (Ellard-Ivey and Douglas 1996), poplar

22

21

20

19/ 19a

18/ 18a

17

16/ 16a

15

Cinnamyl Alcohol Dehydrogenase (CAD)

Hydroxycinnamoyl CoA:Shikimate/ Quinate hydroxycinnamoyltransferase (CST/QST) Cinnamoyl-CoA Reductase (CCR)

Caffeoyl-CoA O-Methyltransferase (CoAOMT) Ferulate-5-Hydroxylase (F5H) (CYP84A) Caffeic Acid/5-Hydroxyferulic acid O-Methyltransferase (COMT)

1.1.1.195

1.2.1.44

2.3.1.133

2.1.1.68

1.14.13.XX

2.1.1.104

1.14.13.36

6.2.1.12

1.14.13.11

143

Cinnamate-4-Hydroxylase (C4H) (CYP73A) 4-Coumaroyl-CoA Ligase (4CL) Coumarate-3-Hydroxylase (C3H) (CYP98A)

E.C number1

Enzyme name

hydroxycinnamoyl-CoA + NADPH,H+ → hydroxycinnamaldehyde + NADP+ + coenzyme A hydroxycinnamaldehyde + NADPH,H+ → hydroxycinnamyl alcohol + NADP+

cinnamate + NADPH,H+ + O2 → p-coumarate + NADP+ + H2 O p-coumarate + coenzyme A + ATP → p-coumaroyl-CoA + AMP + PPi p-coumaroyl-CoA[quinate/shikimate] + NADPH,H+ + O2 → caffeoyl-CoA[quinate/shikimate] + NADP+ + H2 O caffeoyl-CoA + S-adenosyl-L-methionine → feruloyl-CoA + S-adenosyl-Lhomocysteine ferulate + NADPH,H+ + O2 → 5-hydroxyferulate + NADP+ + H2 O caffeate/5-hydroxyferulate+S-adenosylL-methionine → ferulate/sinapate+S-adenosyl-Lhomocysteine p-coumaroyl-CoA + quinate/shikimate → p-coumaroyl-quinate/shikimate + coenzyme A

Reaction catalyzed

At1g72680, At3g19450, At4g34230

At1g15950, At1g80820

At5g48930

At5g54160

At4g36220, At5g04330

At1g24735, At1g67980, At1g67990, At3g61990, At4g26220, At4g34050

At2g30490, At2g30160, At2g30600, At2g30580, At1g51680, At1g65060, At3g21230, At3g21240 At1g74540, At1g74550, At2g40890


Table 9.2 Enzymes of phenylpropanoid metabolism leading to hydroxycinnamic acids and their conjugates, and their corresponding genes in Arabidopsis

9 Wound-Induced Phenylpropanoid Metabolism 201

2.3.1.99

2.1.1.XX

1.14.13.XX

caffeoyltyramine + S-adenosyl-L-methionine → feruloyltyramine+S-adenosyl-Lhomocysteine caffeoyl-CoA + quinate → caffeoylquinate + coenzyme A

Unknown in Arabidopsis




hydroxycinnamoyl-CoA + tyramine → hydroxycinnamoyltyramine + coenzyme A p-coumaroyltyramine + NADPH,H+ + O2 → caffeoyltyramine + NADP+ + H2 O


Reaction catalyzed

2


1

26

25

24

2.3.1.XX

23

Hydroxycinnamoyl CoA:Tyramine hydroxycinnamoyltransferase p-Coumaroyltyramine-3hydroxylase (CT3H) Caffeoyltyramine O-Methyltransferase (CTOMT) Caffeoyl CoA:Quinate caffeoyltransferase (CQC)

E.C number1

Enzyme name


202 M.A. Bernards, L. B˚astrup-Spohr

203

Fig. 9.4 (continued)


204


Fig. 9.4 (continued) Biosynthesis of flavonoids, stilbenes and condensed tannins. The biosynthesis of flavonoids, stilbenes, and condensed tannins is depicted. Cofactors and co-substrates are not shown for clarity. Refer to Table 9.3. Bold arrows indicate steps for which wounding and/or herbivory have been shown to result in induced gene expression and/or enzyme activity. Enzymes are: (14) cinnamate-4-hydroxylase; (15) 4-coumaroyl-CoA ligase; (27) stilbene synthase; (28) chalcone synthase; (29) chalcone isomerase; (30) flavonone 3-hydroxylase; (31) flavonoid 3 -hydroxylase; (32) flavonol synthase; (33) dihydroflavonol 4-reductase; (34) leucoanthocyanidin reductase; (35) anthocyanidin synthase (leucoanthocyanidin dioxygenase). (Adapted in part from Strack 1997; Croteau et al. 2000)

(Peters and Constabel 2002), and at the enzyme level in potato tubers (Bernards et al. 2000). Coumarate-3-hydroxylase (C3H; CYP98A) catalyses the hydroxylation of pcoumarate to form the caffeoyl (o-dihydroxy) ring substitution common in many phenolics. As with 4CL, C3H transcripts are induced by wounding, weevil damage, and/or budworm damage in Sitka spruce (Ralph et al. 2006), and are coordinately upregulated during lignification (Ehlting et al. 2005). Subsequent methylation (caffeoyl-CoA-O-methyltransferase; CCoAOMT and/or caffeic acid/5- hydroxyferulic acid O-methyltransferase; COMT) and 5-hydroxylation (ferulate-5-hydroxylase; F5H; CYP84A) to yield guaiacyl- and syringyl-substituted hydroxycinnamates follow similar expression patterns after wounding and insect damage (Reymond et al. 2004; Ralph et al. 2006), or during development (Ehlting et al. 2005). Consequently, the formation of phenylpropanoids with typical ring substitution patterns is readily achieved in response to mechanical wounding and chewing insect damage to plant tissue, in a coordinate fashion. However, with the exception of some plantinsect interactions (e.g., Ding et al. 2000; Santiago et al. 2005, 2006), hydroxycinnamic acids themselves do not generally accumulate in response to insect damage or wounding, but rather act as precursors to the other phenylpropanoid derivatives that do. And it is in this regard that species-specific end products, and the unique metabolism leading to them, differentiates between responses of different plants to environmental stimuli, including insect damage. The formation of physical barriers such as lignin or suberin in response to wounding is well documented (e.g., Bernards and Lewis 1998; Ginzberg this volume). One of the critical steps committing carbon skeletons to the formation of monolignols (essential building blocks for both lignin and suberin) is catalyzed by the enzyme cinnamoyl-CoA oxidoreductase (CCR), which reduces hydroxycinnamoylCoA derivatives of hydroxycinnamic acids to their corresponding aldehydes (Fig. 9.3, Table 9.2). And while CCR is coordinately up-regulated with other genes of the early steps of phenylpropanoid metabolism during lignification (Ehlting et al. 2005), it appears to be down-regulated upon weevil and/or budworm herbivory (Ralph et al. 2006). This apparent contradiction represents an important example where wounding and herbivory-induced tissue damage are not equivalent. Instead, the hydroxycinnamates generated in Sitka spruce by the up-regulation of genes of the early steps in the phenylpropanoid pathway appear to be channeled into flavonoids (see below). Mechanical wounding results in the accumulation of myriad hydroxycinnamoyl conjugates such as chlorogenic acid (Cottle and Kolattukudy 1982; Razem and


205

Bernards 2002) and hydroxycinnamoyl amides (Malmberg 1984; Razem and Bernards 2002) in potatoes and tomatoes (Pearce et al. 1998). With respect to hydroxycinnamoyltyramines, a hydroxycinnamoyl CoA:tyramine hydroxycinnamoyltransferase (HTH) enzyme has been shown to be induced by wounding (Negrel et al. 1995), which utilizes either p-coumaroyl-CoA or feruloyl-CoA as hydroxycinnamoyl donor (Fig. 9.3). Interestingly, even though feruloyltyramine has been shown to be incorporated into the phenolic domain of wound suberin (Negrel et al. 1996), it is the p-coumaroyl derivative that accumulates when poly(phenolic) domain formation is inhibited (Razem and Bernards 2002). Subsequently, a unique coumarate-3-hydroxylase (C3H) that utilizes p-coumaroyltyramine as substrate in wound-healing potato tubers has been measured in vitro (Pabani and Bernards, unpublished results). This result serves to emphasize the central role of p-coumaroylCoA in hydroxycinnamic acid metabolism as well as the power of using woundinduced (i.e., either mechanical of herbivory associated) tissues as a source of discovering novel biochemistry.

9.3.3 Flavonoids and Tannins Flavonoids are compounds with a C15 aglycone skeleton and form the building blocks of the condensed tannins (Fig. 9.4, Table 9.3). The first compound in the pathway, naringenin chalcone (4,2,4 ,6 -tetrahydroxychalcone), is formed by the condensation of p-coumaroyl-CoA with three molecules of malonyl-CoA, catalyzed by the enzyme chalcone synthase (CHS). Chalcone synthase is considered to be the rate limiting enzyme in the flavonoid pathway (Strack 1997), and has been shown to be induced at both the protein and transcript level by wounding of Norway spruce (Brignolas et al. 1995) and white spruce (Richard et al. 2000), respectively, as well as at the transcript level by wounding and weevil and/or budworm herbivory of Sitka spruce (Ralph et al. 2006). Interestingly, stilbene synthase, which catalyzes the same condensation reaction as CHS, but results in a different folding and cyclyzation sequence to generate stilbenoids (e.g., pinosylvin; Fig. 9.4), is also induced at the protein level by wounding in Norway spruce (Brignolas et al. 1995) and at transcript level by wounding and weevil and/or budworm herbivory of Sitka spruce (Ralph et al. 2006). Presumably the combined synthesis of stilbenes and condensed tannins (from CHS-derived flavonoids) represents a concerted response to both deter insects and prevent opportunistic pathogens from establishing themselves in wounded tissues. Naringenin chalcone is converted into naringenin via a stereospecifc ring closure and isomerization step catalyzed by the enzyme chalcone isomerase (CHI). Chalcone isomerase transcription is induced by wounding and weevil and/or budworm herbivory of Sitka spruce (Ralph et al. 2006). The metabolic fate of naringenin is an important branchpoint in flavonoid biosynthesis, giving rise to the flavonols (e.g., kaempferol, quercitin), dihydroflavonols (e.g., dihydrokaempferol, dihydroquercitin), isoflavonoids (e.g., genistein) and flavones (e.g., apigenin). Collectively, these compounds are referred to as flavonoids. However, while flavonoids themselves have been reported to have deterring effects on insect herbivores (e.g.,

Chalcone Isomerase CHI) Flavanone 3-Hydroxylase (FHT)

29 30

Dihydroflavonol 4-Reductase (DFR) Leucoanthocyanidin Reductase (LAR) Anthocyanidin Synthase (ANS) (Leucoanthocyanidin Dioxygenase) 1.14.11.19

1.17.1.3

1.1.1.219

1.14.11.23

1.14.13.21

5.5.1.6 1.14.11.9


cinnamoyl-CoA + 3 malonyl-CoA → pinosylvin + 4 coenzyme A + 4CO2 p-coumaroyl-CoA + 3 malonyl-CoA → naringenin chalcone + 4 coenzyme A + 3CO2 naringenin chalcone → naringenin naringenin + 2-oxoglutarate + O2 → dihydrokaempferol + succinate + CO2 + H2 O dihydrokaempferol+NADPH,H+ +O2 → dihydroquercitin + NADP+ + H2 O dihydrokaempferol/ dihydroquercitin+2-oxoglutarate+O2 → kaempferol/quercitin + succinate + CO2 + H2 O quercitin + NADPH,H+ → leucocyanidin + NADP+ leucocyanidin + NADPH,H+ → catechin + NADP+ + H2 O leuco(antho)cyanidin + 2oxoglutarate + O2 → anthocyanidin+succinate+CO 2 +2H2 O At2g38240, At4g22880

At1g61720

At5g42800

At1g49390, At2g44800, At3g19010, At3g50210, At5g08640, At5g43935, At5g63580, At5g63590, At5g63595, At5g63600

At5g07990

At1g53520, At5g05270, At3g55120, At5g66220 At1g78550, At3g19000, At3g51240, At4g10490, At4g16330, At5g24530

At1g02050, At4g00040, At4g34850, At5g13930


Reaction catalyzed

2


1

35

34

33

32

Flavonoid 3 -Hydroxylase (F3H) Flavonol Synthase (FLS)

Chalcone Synthase (CHS)

28

31

2.3.1.146

Stilbene Synthase (STS)

273 2.3.1.74

E.C number1

Enzyme name

Table 9.3 Selected enzymes of flavonoids and stilbene synthesis, and their corresponding genes in Arabidopsis

206 M.A. Bernards, L. B˚astrup-Spohr


207

Treutter 2005), they are not as prominent as the condensed tannins with respect to plant-insect interactions. Condensed tannins, also called proanthocyanindins, are formed from the covalent linking of flavan-4-ols (e.g., catechin) and/or flavan-3,4-diols (Fig. 9.4). In Sitka spruce, transcripts for several key steps in the formation of (+)-catechin (the major condensed tannin monomer) are induced several fold by wounding and weevil and/or budworm herbivory (Ralph et al. 2006). These include flavonoid 3 hydroxylase (a CYP75B1 enzyme that hydroxylates dihydrokaempferol to yield dihydroquercitin) and dihydroflavonol 4-reductase. This latter gene, which codes for the enzyme that catalyzes the reduction of the 4 -ketone of the dihydroflavonol ring structure to yield the flavan-3,4-diol leucocyanidin, represents a key step in the formation of condensed tannins. In an earlier report, Peters and Constabel (2002) showed that (in addition to the accumulation of condensed tannins) herbivory induced the expression of this enzyme. Dihydroflavonol 4-reductase catalyzes the penultimate step in the pathway to condensed tannins; the last known step, which converts leuco(antho)cyanidin to (+)-catechin is catalyzed by leucoanthocyanidin reductase. There are no reports of this enzyme being induced by either wounding or herbivory. Similarly, the key step converting naringenin to dihydrokaempferol, catalyzed by flavonone 3-hydroxylase (a 2-oxoglutarate-dependent dioxygenase) does not appear to be induced. It may be that there is sufficient constitutive activity of these key enzymes, the latter of which is also important in the formation of other flavonoids, to support wound/herbivory-induced accumulation. Interestingly, in the Sitka spruce response to wounding, weevil, and budworm damage, the anthocyanidin synthase (leucoanthocyanidin dioxygenase) gene is also induced (Ralph et al. 2006). The enzyme product of this gene catalyses the first step in the formation of anthocyanidins from leucocyanidin, and therefore represents an alternative fate for the carbon skeletons otherwise destined for tannin biosynthesis. The formation of condensed tannins from (+)-catechin and other flavan-4-ols and flavan-3,4-diols remains uncharacterized. It is interesting to note, however, that a number of laccase/diphenol oxidase genes are also induced by wounding and herbivory in Sitka spruce (Ralph et al. 2006); however, whether they are involved in tannin formation remains unknown. It seems clear from the work of Peters and Constabel (2002) and more recently by Ralph et al. (2006) that there is a concerted, coordinate induction of many genes associated with condensed tannin formation, in response to wounding and herbivory, at least in tree species that normally form condensed tannins as defense compounds.

9.4 Concluding Remarks and Perspectives In the preceding sections, the induction of specific subclasses of phenylpropanoid metabolites (hydroxycinnamates and their conjugates; flavonoids; condensed tannins) were described. This treatment of induced phenylpropanoid metabolism, at

208


the transcript and/or enzyme level, is by no means exhaustive. Indeed, a number of other plant/herbivore interactions involving other types of phenylpropanoids have been described, including furanocoumarins in wild parsnip and parsely (e.g., Zangerl 1990, 1999; Lois 1991; Olson and Roseland 1991; Berenbaum and Zangerl 1996), benzoic acids in Nicotiana species (Spencer and Towers 1991; Wildermuth 2006), and hydrolysable tannins in red oak (Schultz and Baldwin 1982). These represent other plant/herbivore specific phenomena that could also provide insights into how plants respond to this type of environmental stress. Nevertheless, the literature describing hydroxycinnamates (and their conjugates), flavonoids, and condensed tannins vis a vis their roles in plant/herbivore interactions may serve as model, providing some general points. First, it is clear that the biosynthesis of phenylalanine, a necessary precursor to most phenylpropanoids, is induced by herbivore damage, presumably along with elements of a nitrogen recycling system that minimizes the energetic impact of using this amino acid as a precursor. Second, there is a coordinate induction of transcripts/enzymes associated with hydroxycinnamic acid formation. These too are essential as precursors to the more specific phenylpropanoids that define different plant-herbivore interactions. Third, there is a coordinate induction of several genes in the flavonoid pathway, leading to the accumulation of flavonoids as well as providing precursors for the condensed tannins. Clearly the literature describing phenylpropanoid metabolism induced by herbivory is incomplete. Recent microarray (genome scale) studies (Reymond et al. 2004; Ehlting et al. 2005; Ralph et al. 2006) have provided evidence for the involvement of many genes in the response of plants to herbivory, including confirmation of the involvement of many expected ones. The next step will certainly involve the functional characterization of these genes, including their selective knock-down and cloning. Forward progress is dependent on expanding analyses to more plant and insect species, as well as monitoring plant reactions to herbivory (or wounding) over relevant time frames. In addition, much needs to be done to advance from our current state of knowledge, i.e., of which genes are transcribed in response to herbivory, to knowledge of how the gene products are assembled into functional metabolic pathways (e.g., post-transcriptional and post-translational modifications, activators, inhibitors, etc.). The study of wound and/or herbivory induced phenylpropanoid metabolism also provides the opportunity to discover novel biochemistry. Defining the phenylpropanoid metabolism of specific plant-insect interactions, both at the transcript and enzyme levels, will lead to the discovery of new pathways, or at least variations on known pathways. It is in defining plant/herbivore-specific variations in phenylpropanoid metabolism that we will discover the underlying strategies used by plants to defend themselves, as well as gain insights into the role(s) of phenylpropanoids in these processes. Acknowledgments The authors are indebted to Dr. S.E. Kohalmi for thoughtful reading of the manuscript. Work conducted in MAB’s lab was supported by an NSERC Discovery Grant.


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Chapter 10

Defense by Pyrrolizidine Alkaloids: Developed by Plants and Recruited by Insects Thomas Hartmann and Dietrich Ober

Pyrrolizidine alkaloids are constitutively expressed toxic plant secondary compounds with sporadic occurrence in distantly related angiosperm families. Physiological and ecological aspects of their role in defense against herbivores are discussed. In Senecio species senecionine N-oxide is synthesized as backbone structure in roots, distributed through the phloem all over the plant and diversified by peripheral reactions yielding the species-specific alkaloid profiles. Except structural diversification the alkaloids do not exhibit any turnover or degradation. They are, however, spatially mobile, slowly allocated and accumulated at strategic important sites of defense, e.g. inflorescences and epidermal tissues. Based on the molecular evolution of the first pathway-specific enzyme, evidence is presented that the pathways of pyrrolizidine alkaloids evolved independently in the various angiosperm taxa. Pyrrolizidine alkaloids are the best studied example for a plant defense system recruited by herbivorous insects. Various adapted insects sequester the alkaloids and utilize them for their own protection against predators and parasitoids. Adapted arctiids (Lepidoptera) are chosen to illustrate the specific integration of a plant defense system into an insect’s biology. This integration concerns sensory recognition and efficient exploitation of plant alkaloid sources as well as maintenance and allocation of the alkaloids in a non-toxic and metabolically safe state. These biochemical adaptations assure the unique role of the alkaloids in the insect’s behavior, for instance, male-to-female transfer of the alkaloids that guarantees an efficient protection of the eggs by coating with alkaloids from both parents.

10.1 Introduction Pyrrolizidine alkaloids (PAs) are a class of constitutively expressed plant defense compounds against herbivores (Hartmann and Witte 1995; Hartmann and Ober 2000). Why should a constitutive defense be considered in a book dedicated to T. Hartmann Institute of Pharmaceutical Biology, Technical University of Braunschweig, D-38106 Braunschweig, Germany e-mail: [email protected]


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T. Hartmann, D. Ober

induced plant defenses? There are good reasons to do so. Firstly, for many chemical defenses the borders between constitutive and induced defenses are floating. Terpenoids (see Bohlmann this volume), phenolics (see Bernards and B˚astrup-Spohr this volume), and even alkaloids such as nicotine (see Steppuhn and Baldwin this volume) are generally expressed constitutively at a certain level which under herbivore pressure can be boosted by induced synthesis. Secondly, in comparison to preformed defenses, induced defenses are less expensive due to lower metabolic costs and allow the focused accumulation of high levels of defense compounds. Constitutive defenses are likely to be more ancient, and provided the basis for the evolution of efficiently regulated inducible defenses. The survival of an herbivorous insect depends greatly on its ability to cope with the plant anti-herbivore endowments. For an herbivore switching to a new potential food-plant it should be easier to become adapted to a preformed defense than to an inducible one. Some herbivores that successfully adapted to a constitutive plant defense even adopted the plant defense system for their own benefit. The mechanistic background of such insect–plant adaptations provides fascinating insights into the abilities of insects to integrate plant-derived defense devices into their own biology. In the first part we review the present knowledge of plant PAs as defense compounds that evolved probably independently in different angiosperm taxa. The second part deals with specialized herbivorous insects that independently acquired the ability to sequester plant PAs. It will be shown that the acquisition of plant PAs may greatly affect the biology of the sequestering insect herbivore.

10.2 Plant Pyrrolizidine Alkaloids 10.2.1 Occurrence and Structural Types The occurrence of PAs is restricted to the angiosperms. They are found scattered and often sporadically in distantly related families. Well known sources are the tribes Senecioneae and Eupatorieae of the Asteraceae, and many genera of the Boraginaceae (Hartmann and Witte 1995). In the Fabaceae, PAs are only found in the tribe Crotalariae where they substitute the quinolizidine alkaloids (Wink and Mohamed 2003). Within the Apocynaceae and Orchidaceae, few genera are known to contain PAs (Hartmann and Witte 1995) while in the Convolvulaceae PAs are only found in a few related species (Jenett-Siems et al. 2005). PAs encompass a rich diversity of about 400 compounds. Despite their diversity they all have one feature in common, they are ester alkaloids composed of a necine base esterified to one or more necic acids (Fig. 10.1). All necine bases are derivatives of the unique bicyclic aminoalcohol, 1-hydroxymethylpyrrolizidine. The necic acids represent a variety of often rather complex branched-chain aliphatic and less abundant aromatic acids. The majority of known PAs can be attributed either to the senecionine type (Fig. 10.1B) or the lycopsamine type (Fig. 10.1C). The former are found mainly in the Asteraceae, tribe Senecioneae, the latter type in three plant

10 Defense by Pyrrolizidine Alkaloids: Developed by Plants and Recruited by Insects HO

HO

A

O

O O

O

O 7

R

O

reduction (spontaneous) 2

necic acid

O

O

9 1

H N

H necine base

N

N-oxidation (enzymatic)

O PA N-oxide non-toxic (hydrophilic)

B

215

PA free base pro-toxic (lipophilic)

SENECIONINE TYPE: Asteraceae (Senecioneae) & Fabaceae OH

HO HO

HO O

O

O

O

O

O O

O

H

O O

O

O

O

O

H

O O

O

H N

N

N

retronecine

senecionine

C

N

retronecine

platyphylline

triangularine

otonecine

platynecine

senkirkine

LYCOPSAMINE TYPE: Asteraceae (Eupatorieae), Boraginaceae & Apocynaceae HO HO OH OH

O

HO H

H

O N

O

N

retronecine

E

H N

N

supinidine

HO O H

H

retronecine

parsonsine

F

IPANGULINE TYPE: Convolvulaceae (Ipomoea)

OH

O

O

HO O

O

O

H O

O N

O

O

OH

O O

O

amabiline

PHALAENOPSINE TYPE: Orchidaceae

O O O H

H

O

heliotridine

O O

OH OH

O

H

7-angeloylechinatine

MONOCROTALINE TYPE: Fabaceae OH

O

O H

O

lycopsamine

D

OH OH

retronecine

monocrotaline

N

trachelanthamidine

T-phalaenopsine

N

OAc

platynecine

ipanguline B

Fig. 10.1 Major structural types of naturally occurring plant pyrrolizidine alkaloids (PAs). A: In many plants 1,2-unsaturated PAs are present as polar, non-toxic N-oxides which are easily reduced producing the respective pro-toxic free base. B: The senecionine type represents the major class of macrocyclic PAs. C: the lycopsamine type represents the major class of monoesters and open-chain diesters. D: The monocrotaline type occurs only in Fabaceae. E and F: The phalaenopsine and ipangularine types represent 1,2-saturated PAs

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taxa, the Asteraceae, tribe Eupatorieae, the Boraginaceae, and the Apocynaceae. PAs of the senecionine type represent typically macrocyclic diesters occasionally accompanied by related open-chain diesters such as triangularine (Fig. 10.1B). PAs of the lycopsamine type are open-chain monoesters or diesters containing at least one unit of 2-isopropyl-2,3-dihydroxybutyrate, the unique C7 -necic acid, which in nature occurs almost exclusively in PAs. In the Apocynaceae, these PAs are occasionally found as macrocyclic triesters such as parsonsine (Fig. 10.1C). In both types of PAs, retronecine is the prevailing necine base but occasionally replaced by other necines. Another unique feature of PAs is that at least in the Asteraceae, Boraginaceae, and Fabaceae they are found as polar N-oxides which, however, are easily spontaneously reduced to the respective free bases (Hartmann and Toppel 1987). Only otonecine esters like senkirkine (Fig. 10.1B) which cannot be N-oxidized are genuinely present as free bases. Species of the Apocynaceae appear to contain PAs only as free bases. Orchidaceae and Convolvulaceae contain only PAs with 1,2saturated necine bases (Fig. 10.1E and F). These PAs are either found as free bases like in the Convolvulaceae (Jenett-Siems et al. 1998), or as mixtures of free bases and their N-oxides like in the orchids (Frölich et al. 2006).

10.2.2 Toxicity PA-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans (Cheeke 1998; Prakash et al. 1999; Stegelmeier et al. 1999). Toxicity induced by PAs has been studied most intensively in vertebrates. Only PAs with a 1,2-double bond in the necine base moiety, e.g. retronecine, heliotridine, supinidine, otonecine (Fig. 10.1) are potentially toxic, but they require metabolic activation to exert toxicity. There are three principle metabolic pathways for the metabolism of 1,2-unsaturated PAs in vertebrates (Fu et al. 2004; Fig. 10.2): (i) hydrolysis of the ester bonds yielding the free necine base; (ii) N-oxidation of the free base, and (iii) oxidation of the necine base moiety to the corresponding dehydropyrrolizidine (pyrrolic ester) derivative. Hydrolysis and N-oxidation are considered as detoxification pathways whereas the formation of a dehydropyrrolizidine creates reactive intermediates that easily form adducts with biological nucleophiles resulting in severe cell toxicity (Fig. 10.2). In vertebrates, this bioactivation is mediated by hepatic cytochrome P450 monooxygenases, particularly CYP3A and CYP2B isoforms (Huan et al. 1998a). Multisubstrate flavine monooxygenases (Huan et al. 1998b) and to a lesser extent P450 enzymes (Williams et al. 1989a, b) are involved in the detoxification of PAs to the corresponding N-oxides. In herbivorous insects, bioactivation of PAs comparable to that of vertebrates exists, since insects possess a rich range of P450 enzymes involved in xenobiotic metabolism (Brattsten 1992; Glendinning and Slansky 1995; Scott et al. 1998; Glendinning 2002). Mutagenic effects of PAs have been demonstrated in Drosophila (Frei et al. 1992), and acute toxicity in larval development of Philosamia ricini (Saturniidae; Narberhaus et al. 2005).

10 Defense by Pyrrolizidine Alkaloids: Developed by Plants and Recruited by Insects

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Fig. 10.2 Detoxification of pro-toxic 1,2-unsaturated pyrrolizidine alkaloids in vertebrates and their conversion into toxic pyrrolic intermediates by cytochrom P450-mediated bioactivation

Many plants store their PAs as non-toxic N-oxides (see Section 5.2.1). However, any herbivorous vertebrate or insect feeding on these plants absorb the PAs as pro-toxic free base since in the reducing gut milieu any ingested N-oxide is easily converted into the respective free base (Mattocks 1986; Lindigkeit et al. 1997). Thus, for a non-adapted herbivore, both forms are pro-toxic. PAs that lack the 1,2-double-bond are not bioactivated (Fu et al. 2004) and therefore in the above discussed sense are non-toxic.

10.2.3 Features of a Constitutive Chemical Defense: Physiological Aspects The basic requirements for a constitutive defense are simple: The defense compound(s) should accumulate stably in the target tissues at concentrations needed to prevent damage by an antagonist, e.g. an insect herbivore. PAs fulfill these requirements in general: they are accumulated in sufficient quantities and are metabolically stable, i.e. they do not show turnover or degradation (Hartmann and Dierich 1998; Frölich et al. 2007). However, PAs are spatially mobile and carried from their sites of synthesis to their sites of accumulation (Hartmann et al. 1989) where they are concentrated within cellular vacuoles (Ehmke et al. 1988). These metabolic characteristics have been established for Senecio species which synthesize senecionine N-oxide as PA backbone structure in the roots. Senecionine N-oxide is distributed through the phloem all over the plant, and is chemically diversified (see below) to

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produce the species-specific PA profiles. Within roots, PA biosynthesis is restricted to specialized cells, as documented by immunolocalization of homospermidine synthase a key enzyme of PA biosynthesis. In Senecio, these cells comprise groups of endodermis cells and adjacent cells of the cortex parenchyma directly opposite to the phloem (Moll et al. 2002). In other PA-producing species the sites of PA synthesis may be different. In Eupatorium, PA biosynthesis occurs in all cells of the root cortex parenchyma (Anke et al. 2004), in Phalaenopsis (Orchidaceae), PA synthesis is restricted to the tips of the aerial roots (Frölich et al. 2006). In the Boraginaceae the site of synthesis may vary from species to species, e.g. only roots (Symphytum officinale), only shoots (Heliotropium indicum), or shoots and roots (Cynoglossum officinale; van Dam et al. 1995b; Frölich et al. 2007). This indicates that for a constitutive defense compound not the site of synthesis per se but the site of accumulation is of ultimate importance. Like for other secondary compounds, chemical diversity and variability are typical features also of PAs. Each species is characterized by its unique PA profile, and great qualitative and quantitative variability is frequently observed between populations. Well studied examples are populations of Senecio jacobaea and Senecio erucifolius (Witte et al. 1992; Vrieling and de Boer 1999), and Senecio vulgaris and Senecio vernalis (von Borstel et al. 1989). Structural diversification has been studied using five Senecio species which differ in their PA profiles (Hartmann and Dierich 1998). All species synthesize senecionine N-oxide as backbone structure which is diversified by simple one or two-step transformations (Fig. 10.3). These reactions affect mainly the necic acid moiety; they include dehydrogenations, various position-specific hydroxylations and epoxidations, and O-acetylations. These ‘peripheral reactions’ proceed slowly and often differ in efficiency between plant organs. Therefore, the PA profile of an individual plant is the result of a dynamic equilibrium of a number of interfering processes such as the rate of supply with de-novo-synthesized senecionine N-oxide, specificity and efficiency of the transformations, the velocity of allocation and, finally, the tissue- and cell-specific storage. Any genetic alteration affecting the efficiency of one of the ‘peripheral reactions’ would drastically modify the PA profile without affecting its overall quantity. Total

Fig. 10.3 Diversification of senecionine N-oxide by peripheral one-step or two-step reactions producing the various species-specific PA-profiles in Senecio species


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plant PA quantity is determined exclusively by the rate of root-specific formation of senecionine N-oxide. This mechanistic scenario explaining the great variation of PA profiles between populations is supported by other studies. A genetic analysis of S. jacobaea populations (Vrieling et al. 1993) revealed that the large phenotypic variation in both total PA content and the qualitative PA profiles is largely due to genetic variation. A comparative analysis of the PA profiles of 24 species of the Senecio section Jacobaea based on a maximum parsimony cladogram inferred from DNA sequence and morphological data (Pelser et al. 2005) resulted in a largely incidental distribution of 26 biogenetically related PAs within the whole clade. Differences in the PA profiles cannot be explained by gain and loss of PA-specific genes during evolution, but rather by a transient switch-off and switch-on of gene expression. Apparently the species are able to randomly shuffle patterns of senecioninederived derivatives from the ‘genetic pool’ of the clade, and thus generating PA profiles of changing diversity and variability.

10.2.4 Features of a Constitutive Defense System: Ecological Aspects As most plant toxins, PAs are bitter tasting for humans and strong feeding deterrents for vertebrates and invertebrates (Boppré 1986). However, direct evidence for their defense function in plants is still sparse. In contrast to an inducible defense where cause (attack) and effect (formation of a defense compound) are obvious, a constitutive defense can only be judged by the absence ore presence of potential herbivores. Therefore, evidence supporting a function of PAs in plant defense is mostly indirect. One line of indirect evidence is tissue-specific accumulation of PAs. Flowers are often the organs showing the highest PA concentrations. This has been demonstrated for Senecio (Hartmann and Zimmer 1986), Heliotropium (Boraginaceae; Frölich et al. 2007), and Phalaenopsis (Orchidaceae; Frölich et al. 2006). In flowering specimens, the amounts of PAs associated with the inflorescences account for 50%–90% of total PAs, indicating an efficient protection of the most valuable reproductive plant organs. In rosette leaves of C. officinale, the youngest leaves contain up to 190 times higher PA levels than older leaves (van Dam et al. 1994). Generalist herbivorous insects only feed on the PA-poor old leaves whereas a specialist herbivore, adapted to PAs, prefers the protein-rich young leaves (van Dam et al. 1995a). In vegetative stems of herbal species like Senecio, PAs accumulate preferentially in the peripheral cell layers (Hartmann et al. 1989), and they are even found in trace amounts on the leaf-surface (Vrieling and Derridj 2003). Again, the functionally valuable young tissues as well as peripheral tissues which provide the first contact for an herbivore are efficiently protected. Another line of indirect evidence indicating a defense role of PAs is the chemical diversity and great variability of the PA profiles. The effect of pure PAs on five generalist insect herbivores revealed that structurally related PAs differ in their deterrent effects and can even act synergistically if applied in mixtures (Macel et al. 2005). This result was taken as argument to emphasize the importance of generalist herbivores on the evolution and maintenance of PA diversity. Further evidence is derived from the

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observation that the geographical distribution of certain S. jacobaea chemotypes is apparently not related to common ancestry but due to similar selection pressure, e.g. herbivores, in a certain region (Macel et al. 2004).

10.2.5 Molecular Evolution of the PA Pathways in Angiosperms All plant PAs share the carbon skeleton of 2-hydroxymethylpyrrolizidine in their necine base moiety (Fig. 10.1). Detailed tracer studies carried out in the late 1970s and 1980s identified the symmetrical polyamine homospermidine as biosynthetic precursor of the necine base moiety (Spenser 1985; Robins 1989). The formation of homospermidine is catalyzed by homospermidine synthase (EC 2.5.1.45, HSS) which was first isolated and characterized from Senecio root cultures (Böttcher et al. 1993, 1994). The enzyme was purified, cloned and functionally expressed in Escherichia coli (Ober and Hartmann 1999). It is encoded by a gene that originated by gene duplication and subsequent diversification from an ancestral gene for deoxyhypusine synthase (EC 2.5.1.46, DHS; Ober and Hartmann 2000). DHS catalyzes the first of two steps in the posttranslational activation of the eukaryotic initiation factor 5A (eIF5A; Caraglia et al. 2001). DHS is a highly conserved enzyme, ubiquitously present in all Eukarya and Archaea. In the activation process of eIF5A, it catalyzes the transfer of the aminobutyl moiety of spermidine to a specific lysine residue of the eIFA precursor protein (Fig. 10.4A). As a side-reaction, DHS is able to catalyze the formation of homospermidine from putrescine (Fig. 10.4A) and thus precisely the main-reaction of HSS. A comparison of the enzymatic and molecular properties of the two enzymes revealed that HSS retained all properties of DHS except the ability to bind the eIF5A precursor protein (Fig. 10.4B; Ober et al. 2003). The evolution of HSS is a conclusive example for evolution by change of function: the duplicate of a ubiquitous gene (DHS) functionally involved in the activation of a regulatory protein was recruited and integrated as HSS into a completely different functional environment, i.e. the biosynthesis of low molecular defense compounds (Fig. 10.4C; Ober and Hartmann 2000; Ober 2005). The confirmed ancestry of HSS from ubiquitously occurring DHS offered the opportunity to address the intriguing question whether the PA pathways within the angiosperms are of monophyletic or polyphyletic origin. Analyzing the pairs of cDNA sequences encoding HSS and DHS from various angiosperm species revealed at least four independent duplication events of the gene encoding DHS resulting in each case in a new gene encoding HSS (Reimann et al. 2005; Fig. 10.5). These duplications occurred early in the evolution of the monocots and of the Boraginaceae, respectively, and even twice within the family of the Asteraceae (i.e., once within the Senecioneae and the Eupatorieae, respectively). Further duplication events are likely in those families with PA-producing species that were not included in this study. These data show that the scattered occurrence of PAs within the angiosperms is a result of convergent evolution. This observation is remarkable against the background that the PA structures produced are often identical. PAs of the lycopsamine type, for example, occur within the tribe Eupatorieae (Asteraceae) and within the


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Fig. 10.4 Origin of homospermidine synthase (HSS) by duplication of the gene encoding deoxyhypusine synthase (DHS). A: DHS catalyzes the aminobutylation of a specific lysine residue of the eIF5A precursor protein (a) and, as side reaction, converts putrescine to homospermidine (b). B: HSS is a DHS that lost the ability to bind the eIF5A precursor protein. Its reactivity and kinetics with putrescine remain unaltered. C: HSS is only known from PA containing plants, it was recruited and integrated into PA biosynthesis and catalyzes the formation of homospermidine, the unique carbon skeleton of the necine bases

families of the Boraginaceae and the Apocynaceae. Despite the independent origin of HSS within these families, the PAs share a number of identical structural features (Fig. 10.1C), such as retronecine as necine base, and the unique C7 -necid acid, 2-isopropyl-2,3-dihydroxybutyrate. Further enzymes of PA biosynthesis will have to be analyzed to get an impression of how the whole pathway was recruited in separate angiosperm lineages.

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Fig. 10.5 Phylogenetic tree of HSS and DHS encoding cDNA sequences of various angiosperm species. Branches of HSS encoding sequences are shown in bold, originating in each case at the duplication event of the DHS-encoding gene (labeled by circles). Modified from Reimann et al. (2004). Species abbreviations: A.t., Arabidopsis thaliana; C.o., Cynoglossum officinale; C.r., Crotalaria retusa; D.c., Dianthus caryophyllus; E.c., Eupatorium cannabinum; H.i., Heliotropium indicum; I.h., Ipomoea hederifolia; L .e., Lycopersicon esculentum; M.a., Musa acuminata; N.t., Nicotiana tabacum; P.h., Petasites hybridus; Phal, Phalaenopsis spec.; S. j., Senecio jacobaea; S.v., Senecio vernalis; S.o., Symphytum officinale

10.3 Utilization of Plant Derived Pyrrolizidine Alkaloids by Insects 10.3.1 Occurence and Distribution of PA Sequestration Among Insects Almost 40 years ago, Miriam Rothschild and her colleagues showed that the larvae of the European cinnabar moth, Tyria jacobaeae (Arctiidae) sequester PAs from their host plant S. jacobaea and maintain the alkaloids through all life stages (Aplin et al. 1968). In the following decades, many further examples of PA-sequestering


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herbivorous insects became known (Hartmann and Witte 1995; Hartmann 1999; Hartmann and Ober 2000). PA sequestration is common within the tiger moth family (Arctiidae; Conner and Weller 2004). About 70 species are known that either sequester PAs with their larval food, like T. jacobaeae, or obtain the alkaloids as adults from dead or withered parts of PA-containing plants. In the latter case, adult moths regurgitate on the plant surface and then reimbibe the fluid containing the dissolved alkaloids (Boppré 1986; Schneider 1987). This behavior in which the plant is only used as ‘drug source’ is called pharmacophagy (Boppré 1984). Pharmacophagy is also common among PA-sequestering butterflies including danaine and ithomiine nymphalid butterflies (Boppré 1990). Pioneering work on PA-sequestration by danaid and ithomiine butterflies has been performed in Australia (Edgar 1982) and Brasil (Brown 1984, 1987; Trigo et al. 1996). Other examples for PA sequestration are found among beetle species. Chrysomelid leaf beetles (Coleoptera) are known for their diverse endogenous chemical defenses. Some beetles possess numerous exocrine glands, from which scores of diminutive droplets of defensive secretions are released when the beetle is attacked. Leaf beetles generally synthesize their own defense compounds, but in some cases, they recruit plant defenses. Species of the palaearctic genus Oreina feeding on PA-containing plants like Adenostyles alliariae (Asteraceae) sequester PAs from their host plants, and concentrate them up to 0.3 mol l−1 in their defense secretions (Pasteels et al. 1988; Hartmann et al. 1997). The same adaptation evolved independently in species of the neotropical genus Platyphora feeding on species containing PAs of the lycopsamine type (Pasteels et al. 2001). Further examples of Coleoptera that sequester plant PAs are flea beetles of the genus Longitarsus feeding on PA-containing species of the Asteraceae and Boraginaceae (Haberer and Dobler 1999; Dobler et al. 2000), and the Brasilian soldier beetle Chauliognathus falax (Cantharidae) feeding on Senecio inflorescences (Klitzke and Trigo 2000). Within the Orthoptera, a single PA-sequestering species was described, the polyphagous grasshopper Zonocerus variegatus (Bernays et al. 1977). Within the Hemiptera, PA-sequestering species are found in three unrelated taxa, including the largid bug Largus rufipennis sucking on Senecio stems (Klitzke and Trigo 2000), and two phloem feeders, i.e. the aphid Aphis jacobaeae feeding on various Senecio species (Witte et al. 1990), and the Mexican scale insect Ceroplastes albolineatus feeding on Pittocaulon (ex Senecio) praecox (Mar´ın Loaiza et al. 2007). All quoted examples for PA sequestration must have evolved independently indicating a significant benefit of PA acquisition for the insects. In most cases, PA sequestration was found to be accompanied by highly specific biochemical adaptations. Leaf beetles, for instance, possess different but specific adaptations to prevent the accumulation of detrimental concentrations of PAs as pro-toxic free bases in metabolically active tissues. They have most efficient membrane carriers which catalyze the transfer of PAs into their defensive glands, and concentrate them in their defense secretions (Hartmann et al. 1999, 2001). In the next chapter, PAsequestering arctiid moths are selected to exemplify the amazingly sophisticated integration of plant-derived PAs into the moth’s biology.

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10.3.2 PA Sequestration by Arctiid Moths 10.3.2.1 Behavioral and Chemo-Ecological Aspects: The Utetheisa ornatrix Story The role of plant-derived PAs in the performance of an insect has most completely been elucidated in U. ornatrix (Eisner et al. 2002). Larvae of this moth obtain their PAs from Crotalaria spp. and retain the alkaloids through metamorphosis. At mating, the male advertises his PA load to the female through the PA-derived male courtship pheromone, hydroxydanaidal. The pheromone is emitted from a pair of androconial scent-brushes (coremata) which the male everts during close-range precopulatory interactions with the female. Females are able to measure the pheromone concentration and differentiate between males that contain unequal pheromone quantities and accept eventually males having higher levels (Conner et al. 1990). During insemination the male transmits a portion of his PAs to the female. These alkaloids are transmitted together with the female’s own load to the eggs during oviposition (Dussourd et al. 1988; Iyengar et al. 2001). PAs protect adults against predation by spiders (Eisner and Eisner 1991; Gonzalez et al. 1999). The PA endowment was also shown to protect the eggs, as the most endangered life stage of the moth, against predation by coccinelid beetles (Dussourd et al. 1988), ants (Hare and Eisner 1993), and chrysopid larvae (Eisner et al. 2000), as well as parasitization by a parasitoid wasp, Trichogramma ostriniae (Bezzerides et al. 2004).

10.3.2.2 Physiological and Biochemical Aspects Arctiids that sequester PAs from their host plants must have acquired at least two novel capabilities: they need to recognize PAs and they must be able to prevent self-poisoning. It is known that PAs are feeding stimulants for PA-adapted arctiids (Boppré 1986; Schneider 1987), but only recently have arctiid caterpillars been shown to possess single sensory neurons in their mouthparts that respond specifically and sensitively (threshold of response 1000 fold molar excess of any of the four tested inhibitors, each of them with different structural folds (Volpicella et al. 2003). On the other hand, also enzymes with differential sensitivity to different inhibitor classes were observed. For instance, a chymotrypsin induced by SKTI was more susceptible to PI-2 than to other inhibitors (Volpicella et al. 2006). Other groups have observed differences in effectiveness of chickpea-derived and potato-derived inhibitors to semi-purified Helicoverpa armigera proteinases (Telang et al. 2005). These data show that a panel of different proteinases can be deployed by adapted pest insects, each with different inhibitor sensitivity. What makes a proteinase insensitive to an inhibitor? The use of synthetic substrates revealed potentially relevant differences between SKTI-induced and constitutive enzymes. Benzoyl-Arg-pNA, which carries a benzoyl group at the P2 position, was digested ten times less efficiently by SKTI-induced trypsin as compared to uninduced trypsin, while substrates with Phe or Arg in the P2 position did not discriminate the two proteinases (Volpicella et al. 2003). This suggests that an important structural feature exists at the S2 site of induced trypsin, which correlates with its resistance to inhibitors. However, the nature of that structural feature cannot be further explored in the absence of a successful recombinant expression system and crystal structures of the proteins. Unequivocal insight in the structural basis of the resistance of insect proteases to plant inhibitors was obtained for the first time from the crystal structure of Helicoverpa zea carboxypeptidase B (Bayes et al. 2005, 2006). This enzyme, referred to as CPBHz, resists inhibition by potato carboxypeptidase inhibitor (PCI), which inhibits all other known enzymes from this class, including Helicoverpa carboxypeptidase A (CPAHa). The CPBHz crystal structure clearly showed that two surface loops of the enzyme assumed an unusual position in comparison to related inhibited enzymes such as CPAHa. In high-affinity complexes, these regions stabilize the interaction between the carboxypeptidase and the inhibitor. This interaction is initially formed by insertion of the flexible C-terminal tail of PCI, which presents

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the P1 and P1’ residue to the active site pocket (Rees and Lipscomb 1982; Molina et al. 1994). However, due to the displacement of these loops on the surface of the enzyme, the inhibitor binds to a more distant position relative to the active site. As a consequence, the P1 and P1’ residues are unable to penetrate the active site deep enough to achieve a strong interaction, and the interaction is easily lost. The evolutionary mechanism by which the proteases evolve towards insensitivity for the inhibitors can be deduced in part from a comparison of the mutated sequence with its closest PI-sensitive homologue. Displacement of protease loops is clearly involved, as is apparent from the CPBHz structure. In one of the two loops, displacement is accompanied by insertion of a residue relative to inhibitor-sensitive enzymes, while in the other loop, a single residue is deleted. In both loops, a the observed number of mutations was higher as compared to other parts of the molecule (Bayes et al. 2005). Interestingly, one could predict that the plant could re-establish inhibition of CPBHz by inserting an additional residue in the substrate-like tail of PCI, thus allowing its P1 residue to reach the active site pocket, but such an inhibitor with an extended tail has not yet been found or evolved.

11.4.2 Inactivation with Specific Proteases Although generally stable against many proteases, some insects have evolved specific proteases capable of effectively degrading plant protease inhibitors. We observed this phenomenon recently with Plutella xylostella degrading MTI-2 when expressed in Arabidopsis (Yang et al. submitted). Others found degradation of dietary protease inhibitors by insects like black vine weevil, Phaedon cochleria and H. armigera (Michaud 1997; Girard et al. 1998; Giri et al. 1998). In our opinion, many cases of specific inactivation may have escaped detection due to the experimental set up chosen by most authors, in which comparing short and prolonged preincubation of the inhibitors with the gut proteases is not standard procedure.

11.5 Perspectives 11.5.1 Regulation is the Key: Did Insects and Plants Both Think of It. . .? The existence of PI-insensitive proteases in the insect gut and their specific upregulation in response to proteinase inhibitors in the diet provides a clear benefit to the insect and is likely monitored by some kind of feedback mechanism which has not yet been elucidated. However, the parallel observation of sensitive enzymes being downregulated could be beneficial to both insects and plants alike. The insects don’t waste their energy on useless (inhibited) proteases, but the plants similarly, if they would govern this process, could spend less energy on synthesizing an effective concentration of inhibitors. Indeed if plants could successfully antagonize

11 Plant Protease Inhibitors

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the regulatory feedback system operational in insect guts, for PI-sensitive and -insensitive proteases alike, they would have found a second way independent of direct inhibition to achieve the same net result: lower protease levels in the gut, leading to slower development and higher mortality. Hijacking the regulation of proteases in the insect gut is, therefore, a holy grail for plants, and preventing them from achieving this goal is the lifeline of the insects: an attractive second stage of evolutionary battle between proteases and inhibitors. However, has the script been written? How likely is it, that plants have evolved such signals for the specific purpose of downregulating proteases in insects? Currently, there are a few peptide hormones known for receptor-mediated up- or downregulation of serine proteases. In mammals the monitor peptide, member of the PSTI gene family, and the luminal cholecystokinin releasing factor LCRF are known to upregulate pancreatic protease secretion by stimulating cholecystokinin release in the blood (Iwai et al. 1988; Spannagel et al. 1996). Excess proteases subsequently degrade the monitor/LCRF peptides and stop the secretion. In mosquitoes after a blood meal the oostatic hormone or trypsin modulating oostatic factor (TMOF) was the only peptide found so far to actively signal the downregulation of trypsin secretion into the gut (Borovsky et al. 1990). Remarkably the TMOF peptide, which consists of only ten amino acids, is also active in the lepidopteran herbivore H. virescens. TMOF actively downregulates proteinase secretion when injected into the hemolymph of the larvae (Nauen et al. 2001), but surprisingly also when it is expressed in plants fused to other peptides, and when it reaches the larvae through the gut after eating the plant leaves. Expressed as a potentially cleavable six-domain repeat, or replacing systemin in prosystemin, or in C-terminal fusion to the coat protein of TMV, TMOF reduced larval development and decreased both gut trypsin and chymotrypsin activity (Tortiglione et al. 2002, 2003; Borovsky et al. 2006). The TMOF peptide presumably signals through the interaction with receptors present on cells lining the gut. Such receptors are also potential targets for plant peptides or proteins to upset gut physiology. This brings us to the last unexplored area of plant protease inhibitors: the function(s) of the N- and C-terminal peptides which extend from most of the basic inhibitor domains that have been characterized in plants. The extensions appear in significant variation, both in length and sequence, within most inhibitor gene families. As an example, Table 11.3 shows the peptides extending from the mature forms of the Bowman–Birk inhibitors found in soybean and peanut. These sequences represent specific insertions and/or deletions. The extensions are not likely to be involved in the subcellular targeting of inhibitors, since only the mature proteins are shown and the targeting peptides are already removed. Therefore, these peptide domains may serve a direct role in plant defense. Potential functions include the modification of the pI of the protein molecule to favor the interaction with the protease, or else, the binding to cellular receptors lining the herbivore gut to modulate or antagonize the regulatory processes of the insect. Understanding the regulation of protease expression in insects and the potential role of plant protease inhibitors to interfere with this regulation promises to be a rewarding research area.

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Table 11.3 Comparison of the N- and C-terminal peptides of the Bowman–Birk inhibitors of soybean and peanut extending outside the basic inhibitory domain for which no function has been assigned Source

Code1

N-terminal sequence3

Inhibitor2

C-terminal sequence3

Soybean Soybean Soybean Soybean Peanut Peanut

MER21210 MER21212 MER19956 MER19957 MER18102 MER18107

SDQSSSYDDDEYSKPCCSDHSSSDDESSKPCCSDHHQHSNDDDSSKPCCSDHHQHSNDDESSKPCCEASSSSDDNVCCAASDCC-

-inhibitor-inhibitor-inhibitor-inhibitor-inhibitor-inhibitor-

-CKSRDD -CKSSDEDDD -CKPSEDDKENY -CKPSQDDKENY -CRS -CA

1

Codes refer to the MEROPS protease inhibitor database: http://merops.sanger.ac.uk/ This represents the conserved two domains involved in protease inhibition 3 The sequences represent the mature stored forms after removal of the signal peptide and other targeting peptides 2

11.5.2 Creative Evolution In this chapter we reviewed the ways in which plants and plant predators have evolved creative solutions in the battle for scarce essential amino acids. We showed that on both the plant and insect side many different gene families are involved with intricate specialized expression patterns among the different members. They provide an evolutionary and ecological genetic fingerprint of their functional role, and, if anything, this chapter highlighted the little that is actually known about the intricate fabric of functional relationships that are still out there to be discovered and admired. Some of the hypotheses we put forward are currently only supported by circumstantial evidence, but provide attractive alleys which functional evolution may have gone. At first glance, this topic of plant–insect/pathogen interactions appears to be only a clash of two protein molecules. However, due to the opportunistic nature of evolution, this battle may have taken on many new appearances. We hope our account of these possibilities provides inspiration to researchers to investigate them. The availability of fully sequenced genomes on both the plant and insect side, and the excellent tools to specifically knock down and overexpress specific genes provide exactly the instruments needed to allow a whole new set of discoveries in this area.

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Beekwilder J, Schipper B, Bakker P, Bosch D, Jongsma M (2000) Characterization of potato proteinase inhibitor II reactive site mutants. Eur J Biochem 267:1975–1984 Berger A, Schechter I (1970) Mapping the active site of papain with the aid of peptide substrates and inhibitors. Philos Trans R Soc Biol Sci 257:249–264 Birk Y, Gertler A, Khalef S (1963) A pure trypsin inhibitor from soya beans. Biochem J 87:281–284 Bode W, Huber R (2000) Structural basis of the endoproteinase–protein inhibitor interaction. Biochim Biophys Acta 1477:241–252 Borovsky D, Carlson DA, Griffin PR, Shabanowitz J, Hunt DF (1990) Mosquito oostatic factor: a novel decapeptide modulating trypsin-like enzyme biosynthesis in the midgut. FASEB J 4:3015–3020 Borovsky D, Rabindran S, Dawson WO, Powell CA, Iannotti DA, Morris TJ, Shabanowitz J, Hunt DF, DeBondt HL, DeLoof A (2006) Expression of Aedes trypsin-modulating oostatic factor on the virion of TMV: a potential larvicide. Proc Natl Acad Sci USA 103:18963–18968 Bowman DE (1946) Differentiation of soy bean antitryptic factors. Proc Soc Exp Biol Med 63:547–550 Bown DP, Wilkinson HS, Gatehouse JA (1997) Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochem Mol Biol 27:625–638 Bown DP, Wilkinson HS, Gatehouse JA (2004) Regulation of expression of genes encoding digestive proteases in the gut of a polyphagous lepidopteran larva in response to dietary protease inhibitors. Physiol Entomol 29:278–290 Brito LO, Lopes AR, Parra JRP, Terra WR, Silva MC (2001) Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases. Comp Biochem Physiol Biochem Mol Biol 128:365–375 Chougule NP, Giri AP, Sainani MN, Gupta VS (2005) Gene expression patterns of Helicoverpa armigera gut proteases. Insect Biochem Mol Biol 35:355–367 Clauss MJ, Mitchell-Olds T (2004) Functional divergence in tandemly duplicated Arabidopsis thaliana trypsin inhibitor genes. Genetics 166:1419–1436 De Leo F, Gallerani R (2002) The mustard trypsin inhibitor 2 affects the fertility of Spodoptera littoralis larvae fed on transgenic plants. Insect Biochem Mol Biol 32:489–496 Ferreira C, Capella AN, Sitnik R, Terra WR (1994) Properties of the digestive enzymes and the permeability of the peritrophic membrane of Spodoptera frugiperda (Lepidoptera) larvae. Comp Biochem Physiol Physiol 107:631–640 Ferry N, Edwards MG, Gatehouse J, Capell T, Christou P, Gatehouse AMR (2006) Transgenic plants for insect pest control: a forward looking scientific perspective. Transgenic Res 15:13–19 Girard C, Le Metayer M, Bonade-Bottino M, Pham-Delegue MH, Jouanin L (1998) High level of resistance to proteinase inhibitors may be conferred by proteolytic cleavage in beetle larvae. Insect Biochem Mol Biol 28:229–237 Giri AP, Harsulkar AM, Deshpande VV, Sainani MN, Gupta VS, Ranjekar PK (1998) Chickpea defensive proteinase inhibitors can be inactivated by podborer gut proteinases. Plant Physiol 116:393–401 Gosalia DN, Salisbury CM, Ellman JA, Diamond SL (2005) High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays. Mol Cell Prot 4:626–636 Green TR, Ryan CA (1972) Wound-induced proteinase inhibitor in plant leaves – possible defense mechanism against insects. Science 175:776–777 Greenblatt HM, Ryan CA, James MN (1989) Structure of the complex of Streptomyces griseus proteinase B and polypeptide chymotrypsin inhibitor-1 from russet burbank potato tubers at ˚ resolution. J Mol Biol 205:201–228 2.1 A Gruden K, Kuipers AGJ, Guncar G, Slapar N, Strukelj B, Jongsma MA (2004) Molecular basis of colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases. Insect Biochem Mol Biol 34:365–375

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Gutierrez-Campos R, Torres-Acosta JA, Saucedo-Arias LJ, Gomez-Lim MA (1999) The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nature Biotech 17:1223–1226 Hejgaard J (2005) Inhibitory plant serpins with a sequence of three glutamine residues in the reactive center. Biol Chem 386:1319–1323 Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 330:160–163 Iwai K, Fushiki T, Fukuoka S (1988) Pancreatic-enzyme secretion mediated by novel peptide: monitor peptide hypothesis. Pancreas 3:720–728 Jongsma MA, Bakker PL, Peters J, Bosch D, Stiekema WJ (1995) Adaptation of Spodoptera exigua larvae to plant proteinase-inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 92:8041–8045 Jongsma MA, Bolter C (1997) The adaptation of insects to plant protease inhibitors. J Insect Physiol 43:885–895 Koepke J, Ermler U, Warkentin E, Wenzl G, Flecker P (2000) Crystal structure of cancer chemo˚ resolution. preventive Bowman–Birk inhibitor in ternary complex with bovine trypsin at 2.3 A Structural basis of janus-sfaced serine protease inhibitor specificity. J Mol Biol 298:477–491 Kunitz M (1946) Crystalline soybean trypsin inhibitor. J Gen Physiol 29:149–154 Laskowski M, Kato I, Ardelt W, Cook J, Denton A, Empie MW, Kohr WJ, Park SJ, Parks K, Schatzley BL, Schoenberger OL, Tashiro M, Vichot G, Whatley HE, Wieczorek A, Wieczorek M (1987) Ovomucoid 3rd domains from 100 avian species – isolation, sequences, and hypervariability of enzyme-inhibitor contact residues. Biochemistry 26:202–221 Liu YL, Salzman RA, Pankiw T, Zhu-Salzman K (2004) Transcriptional regulation in southern corn rootworm larvae challenged by soyacystatin N. Insect Biochem Mol Biol 34:1069–1077 Mazumdar-Leighton S, Broadway RM (2001a) Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor. Insect Biochem Mol Biol 31:633–644 Mazumdar-Leighton S, Broadway RM (2001b) Transcriptional induction of diverse midgut trypsins in larval Agrotis ipsilon and Helicoverpa zea feeding on the soybean trypsin inhibitor. Insect Biochem Mol Biol 31:645–657 Michaud D (1997) Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol 15:4–6 Molina MA, Marino C, Oliva B, Aviles FX, Querol E (1994) C-tail valine is a key residue for stabilization of complex between potato inhibitor and carboxypeptidase-A. J Biol Chem 269:21467–21472 Moon J, Salzman RA, Ahn JE, Koiwa H, Zhu-Salzman K (2004) Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defence protease inhibitor. Insect Mol Biol 13:283–291 Nauen R, Sorge D, Sterner A, Borovsky D (2001) Tmof-like factor controls the biosynthesis of serine proteases in the larval gut of Heliothis virescens. Arch Insect Biochem Physiol 47:169–180 Pereira PJB, Bergner A, Macedo-Ribeiro S, Huber R, Matschiner G, Fritz H, Sommerhoff CP, Bode W (1998) Human ␤-tryptase is a ring-like tetramer with active sites facing a central pore. Nature 392:306–311 Qu LJ, Chen J, Liu MH, Pan NS, Okamoto H, Lin ZZ, Li CY, Li DH, Wang JL, Zhu GF, Zhao X, Chen X, Gu HG, Chen ZL (2003) Molecular cloning and functional analysis of a novel type of Bowman–Birk inhibitor gene family in rice. Plant Physiol 133:560–570 Rawlings ND, Morton FR, Barrett AJ (2006) MEROPS: the peptidase database. Nucl Acids Res 34:D270–D272 Rees DC, Lipscomb WN (1982) Refined crystal-structure of the potato inhibitor complex of carboxypeptidase-A at 2.5-A resolution. J Mol Biol 160:475–498 Ryan CA (1990) Protease inhibitors in plants – genes for improving defenses against insects and pathogens. Ann Rev Phytopathol 28:425–449


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Song HK, Suh SW (1998) Kunitz-type soybean trypsin inhibitor revisited: refined structure of its complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator. J Mol Biol 275:347–363 Spannagel AW, Green GM, Guan DF, Liddle RA, Faull K, Reeve JR (1996) Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion. Proc Natl Acad Sci USA 93:4415–4420 Telang MA, Giri AP, Sainani MN, Gupta VS (2005) Characterization of two midgut proteinases of Helicoverpa armigera and their interaction with proteinase inhibitors. J Insect Physiol 51:513–522 Tortiglione C, Fanti P, Pennacchio F, Malva C, Breuer M, De Loof A, Monti LM, Tremblay E, Rao R (2002) The expression in tobacco plants of Aedes aegypti trypsin modulating oostatic factor (aea-tmof) alters growth and development of the tobacco budworm, Heliothis virescens. Mol Breed 9:159–169 Tortiglione C, Fogliano V, Ferracane R, Fanti P, Pennacchio F, Monti LM, Rao R (2003) An insect peptide engineered into the tomato prosystemin gene is released in transgenic tobacco plants and exerts biological activity. Plant Mol Biol 53:891–902 Volpicella M, Ceci LR, Cordewener J, America T, Gallerani R, Bode W, Jongsma MA, Beekwilder J (2003) Properties of purified gut trypsin from Helicoverpa zea, adapted to proteinase inhibitors. Eur J Biochem 270:10–19 Volpicella M, Ceci LR, Gallerani R, Jongsma MA, Beekwilder J (2001) Functional expression on bacteriophage of the mustard trypsin inhibitor MTI-2. Biochem Biophys Res Commun 280:813–817 Volpicella M, Cordewener J, Jongsma MA, Gallerani R, Ceci LR, Beekwilder J (2006) Identification and characterization of digestive serine proteases from inhibitor-resistant Helicoverpa zea larval midgut. J Chrom B Analyt Technol Biomed Life Sci 833:26–32 Yang L, Fang ZY, Dicke M, van Loon JJA, Jongsma MA (2008) The diamondback moth, Plutella xylostella specifically inactivates Mustard Trypsin Inhibitor 2(MTI2) to overcome host plant defence (submitted) Zavala JA, Patankar AG, Gase K, Hui DQ, Baldwin IT (2004) Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses. Plant Physiol 134:1181–1190 Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) Genevestigator. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–2632

Chapter 12

Defensive Roles of Polyphenol Oxidase in Plants C. Peter Constabel and Raymond Barbehenn

Plant polyphenol oxidases (PPOs) are widely distributed and well-studied oxidative enzymes, and their effects on discoloration in damaged and diseased plant tissues have been known for many years. The discovery in C.A. Ryan’s laboratory in the mid-1990s that tomato PPO is induced by the herbivore defense signals systemin and jasmonate, together with seminal work on PPO’s possible effects on herbivorous insects by G. Felton and S. Duffey has motivated many studies of PPO in the context of plant-herbivore defense. The cloning and characterization of PPO cDNAs from multiple plant species now allows for direct testing of defensive functions of PPO using transgenic plants. These have shown that PPO can contribute to insect herbivore and pathogen resistance, although how this occurs is only now being investigated more closely. Here we review progress in the functional analysis of PPO in plant defense against pests, and describe recent results that address the mechanisms of PPO as an anti-herbivore protein. We suggest that assumptions of how PPO functions as an anti-nutritive defense against lepidopterans needs to be reexamined in light of the near anoxic conditions in lepidopteran midguts. Ultimately, the efficacy of PPO should be directly tested in a greater variety of plant-insect interactions. In addition, the identification of the endogenous PPO substrates will help to define defensive and potentially other roles for PPO in plants.

12.1 Introduction Polyphenol oxidases (PPOs) are ubiquitous copper-containing enzymes which use molecular oxygen to oxidize common ortho-diphenolic compounds such as caffeic acid and catechol to their respective quinones. PPO-generated quinones are highly reactive and may cross-link or alkylate proteins, leading to the commonly observed brown pigments in damaged plant tissues and plant extracts. The conspicuous pigments are generally undesirable in food products, and the role of PPO in browning C.P. Constabel Centre for Forest Biology and Department of Biology, University of Victoria, Victoria, BC, Canada V8W 3N5, e-mail: [email protected] A. Schaller (ed.), Induced Plant Resistance to Herbivory, C Springer Science+Business Media B.V. 2008

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has prompted numerous studies on PPO in food and beverages. In parallel, the potential roles for PPO in plant defense against pests have motivated many studies on PPO in an ecological context, though few of these have used a transgenic approach. Functional and mechanistic studies on PPO in plant-insect interactions using PPOmodified transgenic plants have recently been reported, providing new insight into the biology of this versatile enzyme.

12.2 The Biochemistry of Plant Polyphenol Oxidase Polyphenol oxidase (catechol oxidase; E.C. 1.10.3.2) has been purified and characterized from a wide range of plant species and a variety of tissues (Constabel et al. 1996; Mayer 2006), and activity levels using common substrates vary widely (Constabel and Ryan 1998). Key features of PPOs are two conserved copper-binding domains, and N-terminal chloroplast and thylakoid transit peptides (van Gelder et al. 1997; Marusek et al. 2006). The size of the predicted mature PPO proteins is typically 54–62 kD (Constabel et al. 1996; van Gelder et al. 1997). However, some PPOs are processed further, and in some cases only the processed form is fully active (Rathjen and Robinson 1992). Many PPOs are predicted to contain a proteolytic processing site near the C-terminus of the polypeptide (Marusek et al. 2006). Based on its association with browning reactions in crop plants, PPO has been characterized in a wide variety of food plants including banana, wheat, quince, and avocado, and a number of chemical inhibitors have been identified (reviewed in Mayer 2006; Yoruk and Marshall 2003). Although PPO is found at significant levels in a variety of fruits, vegetables and grains, its biological function in these tissues has rarely been studied. PPO is expressed in many different tissues and organs, including roots, leaves, flowers, and vascular tissue (Constabel et al. 1996). Its presence in chloroplasts led to the proposal that PPO may function in pseudocyclic photophosphorylation or as a modulator of oxygen levels, yet to date little evidence supports these roles (Steffens et al. 1994). A complicating issue regarding PPO function has been the separate localization of its phenolic substrates in plant vacuoles, so that the cell would have to be broken in order for PPO to oxidize phenols; this is most likely to occur following pest or pathogen challenge. Roles of PPO in plant defense are thus commonly discussed, although direct evidence for such roles has become available only recently. A puzzling feature of PPO has been a variable degree of latency, so that PPOs from some species need to be activated with detergents or proteases for full activity. For example, tomato leaf PPO is extracted in its fully active form (Constabel et al. 1995), but poplar leaf PPO requires activation with protease or detergent (Constabel et al. 2000). Recent experiments suggest that such activating treatments function by removal of a peptide from the active site, via proteolysis or partial unfolding of the polypeptide, respectively (Gandia-Herrero et al. 2005). Treatment with low pH can also activate latent PPOs, presumably via a conformational change of the active site, increasing its accessibility to substrates (Kanade et al. 2006).

12 Polyphenol Oxidases in Plant Defense

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PPO latency may be significant for its defensive function; we recently showed that latent poplar PPO is activated by its passage through caterpillar guts (Wang and Constabel 2004). PPOs are known to have broad substrate specificities. Thus, an enzyme from any given source may be capable of oxidizing a variety of simple ortho-diphenolics, such as caffeic acid and its conjugates, catechol derivatives, or dihydroxyphenylalanine (DOPA). However, enzymes from different plant species exhibit distinct preference profiles (reviewed in Constabel et al. 1996). Flavonoids with ortho-dihydroxy phenolic rings have been found to be PPO substrates, for example, catechin, (−)-epicatechin, and myricetin (Guyot et al. 1996; Jimenez and GarciaCarmona 1999). Some reports also describe the oxidation of trihydroxy phenolics, such as gallic acid, by PPO (Shin et al. 1997), but it is not clear how widespread this is. Furthermore, for some PPOs a monophenolase activity has been described (Wuyts et al. 2006). Such enzymes may hydroxylate a monophenol such as tyrosine, which can then undergo further oxidation by the polyphenol oxidase activity to the quinone. Tyrosine hydroxylation by PPO has been described in pokeweed, where this reaction constitutes part of the biosynthetic pathway leading to betalains (Gandia-Herrero et al. 2005). While the range of substrates accepted by isolated PPOs can be readily defined in vitro, knowledge of the substrates that are utilized in planta or during defense reactions is much scarcer. Caffeic acid esters such as chlorogenic acid (caffeoylquinate) are excellent PPO substrates and are very common plant metabolites. In tomato and coffee, chlorogenic acid has been identified as the most likely in vivo PPO substrate (Melo et al. 2006; Li and Steffens 2002). Caffeic acid esters are ubiquitous as lignin precursors (Humphreys and Chapple 2002), but in most species these may not accumulate to sufficient levels to be considered likely PPO substrates. In Populus tremuloides, catechol is postulated to be released by the breakdown of the abundant phenolic glycosides (Clausen et al. 1989), and would therefore be available as a substrate in damaged tissues (Haruta et al. 2001). For most PPOs, however, the endogenous substrates are unknown. The importance of identifying the in planta PPO substrates and understanding the overall phytochemical context of PPO-containing plants is emphasized by recent reports of PPO-like enzymes with biosynthetic roles as hydroxylases of secondary metabolites (Cho et al. 2003; Nakayama et al. 2000). PPO has been extensively studied by biologists, plant pathologists, and ecologists interested in mechanisms of defense against pests and pathogens. Based on the browning reactions resulting from the reactive PPO-generated quinones, PPO has often been suggested to function as a defense against pests and pathogens. A most dramatic illustration of the efficacy of PPO in this context comes from work on Solanum berthaultii in which extremely high PPO levels (45% of soluble protein) are found in glandular trichomes (Kowalski et al. 1992). Breakage of the trichomes by small-bodied insects such as aphids leads to rapid PPO-mediated oxidation and polymerization of phenolics, ultimately entrapping insects, or occluding their mouthparts with a sticky polymer (Kowalski et al. 1992). In most species, however, leaf PPO is found not in trichomes but in mesophyll cells. Here, the PPO-generated

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quinones were proposed to alkylate dietary protein during insect feeding, and to degrade essential amino acids in insect guts (Felton et al. 1989, 1992). PPO-mediated protein alkylation has been demonstrated to operate against lepidopteran pests in the presence of oxygen and in artificial diets, and is now being investigated in more detail in midgut fluids (see Section 12.5 below).

12.3 PPO and Induced Herbivore Defense in Tomato and Other Plants The idea that PPO may act as an anti-nutritive defense against leaf-eating insects was first suggested by G. Felton and S. Duffey, who showed an inverse correlation of Heliothis zea growth and PPO levels in tomato plants (Felton et al. 1989). Strong support for an anti-herbivore role of PPO came from the discovery that the herbivore defense-inducing signal molecules systemin and methyl jasmonate (MeJA) induce PPO activity and PPO mRNA levels in tomato leaves (Constabel et al. 1995). Systemin is a short peptide required for systemic wound signaling in tomato which strongly upregulates tomato herbivore defenses (Ryan 2000; Narváez-Vásquez and Orozco-Cárdenas, this volume). Though no longer considered to be the primary systemic signal, it is required for the generation of such a signal (Schilmiller and Howe 2005). PPO and other defenses were also found to be strongly induced by MeJA and oligogalacturonic acid, major plant defense signaling compounds (Constabel et al. 1995). Since PPO induction in tomato by multiple signals occurs in parallel with a suite of other anti-herbivore proteins including several types of protease inhibitors (PIs) and the anti-nutritive enzymes arginase and threonine deaminase (Bergey et al. 1996; Chen et al. 2005), PPO is thought to play a similar role in defense against insects. In tobacco, PPO and PIs are upregulated by tobacco systemin as well as by MeJA (Constabel and Ryan 1998; Ren and Lu 2006). Likewise, strong herbivore-, wound-, and MeJA-induction of PPO was shown in leaves of several poplar species (Constabel et al. 2000; Haruta et al. 2001). Hybrid poplar (Populus trichocarpa x P. deltoides) has a strong systemic inducible defense response, which also includes trypsin inhibitors and chitinases, both with confirmed anti-insect activities (Parsons et al. 1989; Lawrence and Novak 2001, 2006). Recent large scale genomics experiments have underscored the complexity of the herbivore defense response in hybrid poplar, which involves upregulation of many additional putative defense genes (Christopher et al. 2004; Ralph et al. 2006; Major and Constabel 2006). Overall, the co-induction of PPO with other herbivore defense proteins in several plant species has provided support for its anti-herbivore function. The induction of PPO in tomato by both insects and MeJA has been replicated in both laboratory and field studies (Stout et al. 1998; Thaler et al. 1996, 2002). Furthermore, the early work in tomato stimulated numerous studies on inducible PPO in diverse plant species. The inducibility of PPO by wounding or MeJA treatment has been confirmed in other plants, including both herbaceous crops and trees (Constabel and Ryan 1998). The induction of PPO by herbivory has now been shown


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Table 12.1 Studies of PPO induction in plants by herbivores with unknown effects on herbivores Plant taxa

Insect species (Order)1

Solanaceae Tomato Potato

Leptinotarsa decemlineata (C) ∼ 2X L. decemlineata regurgitant ∼3–7X

Salicaceae Poplar (hybrid)

Malacosoma disstria (L)

∼ 12X

Constabel et al. (2000)

Betulaceae Black alder

Agelastica alni (C)

∼ 3X

Tscharntke et al. (2001)

Poaceae Buffalograss Blissus occiduus (H) Barley, wheat, oats Diuraphis noxia (Ho)

None None

Heng-Moss et al. (2004) Ni et al. (2001)

Fabaceae Common bean Soybean Soybean Soybean

Melanoplus differentialis (O) Helicoverpa zea (L) Ceratoma trifurcata (C) Spissistilus festinus (H)

∼ 2X None None 1.6X

Alba-Meraz and Choe (2002) Bi and Felton (1995) Felton et al. (1994) Felton et al. (1994)

Malvaceae Cotton

Helicoverpa zea (L)

Not detectable Bi et al. (1997)

Theaceae Tea

Helopeltis theivora (H)

∼2–3X

1

Induction

Reference Felton (1992) Kruzmane et al. (2002)

Chakraborty and Chakraborthy (2005)

C = Coleoptera, L = Lepidoptera, H = Heteroptera, Ho = Homoptera, O = Orthoptera.

for a taxonomically diverse group of plants (Table 12.1), and this induction has commonly been interpreted as a direct response against the herbivore. By contrast, studies of the potential impact of induced PPO against the herbivores have largely been done with a more limited range of plants and herbivores, primarily noctuid caterpillars on tomato and other species in the Solanaceae (Table 12.2). The results of 11 of 16 experiments demonstrate PPO induction and are consistent with the hypothesis that induced PPO contributes to defense against herbivores. As with all correlative studies, however, it is not possible to determine the specific impact of induced PPO on herbivores due to the other biochemical changes that occur in damaged plants (e.g., Hermsmeier et al. 2001; Thaler et al. 2001; Chen et al. 2005; Major and Constabel 2006). The direct effects of PPO on insect herbivores can thus best be tested using transgenic plants, where PPO levels are manipulated independently of other traits (see Section 12.4). Other studies of the association between PPO activity and insect herbivore performance using (1) plant genotypes that vary in resistance to herbivory, (2) ontogenetic variation in PPO activity within the plant, and (3) leaves treated with PPO, yielded mixed results. A potato genotype with high PPO activity had increased resistance to the Colorado potato beetle (Leptinotarsa decemlineata; Castanera et al. 1996), while resistance to the coffee leaf miner (Leucoptera coffeella; Diptera) was apparently unaffected by higher levels of PPO in coffee

Epirrita autumnata (L)

Betulaceae Montain birch

2

∼ 3X

∼ 2X ∼ 2X ∼ 3X ∼ 3X ∼ 3X ∼ 3X ∼ 3X ∼ 5X Decreased Decreased ∼ 2X ∼ 4X ∼4–7X Decreased Decreased

Induction

(−)

(−) (−) (0) (−) (−) (−) (0) (−) (+) (0) (−) (−) (−) (0) (0)

Effect

Epirrita autumnata (L) RGR

Spodoptera exigua (L) GR Spider mite (A) numbers Manduca sexta (L) RGR Trichoplusia ni (L) RGR Thrips (T) damage Spider mite (A) numbers Spodoptera exigua (L) RGR Spodoptera exigua (L) RGR Spodoptera exigua (L) RGR Manduca sexta (L) RGR Manduca sexta (L) RGR Field damage by (L) and (O) Leptinotarsa decemlineata (C) RGR Manduca sexta (L) herbivory Epitrix sp. (C) herbivory

Herbivore1 and response2

C = Coleoptera, L = Lepidoptera, O = Orthoptera, T = Thysanoptera, A = Arachnida. GR = growth rate (mg/day), RGR = relative growth rate (mg/mg/day).

Helicoverpa zea (L) Helicoverpa zea (L) Jasmonic acid Jasmonic acid Jasmonic acid Jasmonic acid Jasmonic acid Jasmonic acid Macrosiphum euphorbiae (H) Wind stress Jasmonic acid clipped sagebrush High constitutive PPO High N fertilizer High N fertilizer

Solanaceae Tomato Tomato Tomato Tomato Tomato Tomato Tomato (wild) Tomato Tomato Tomato Tomato Tobacco (wild) Potato (wild) Horsenettle Horsenettle

1

Inducing agent (Order)1

Plant taxa

Table 12.2 Induction of PPOs in plants with effects tested on herbivores

Ruuhola and Yang (2006)

Stout et al. (1998) Stout et al. (1998) Thaler et al. (2002) Thaler et al. (2002) Thaler et al. (1999, 2002) Thaler et al. (2002) Thaler et al. (2002) Thaler et al. (1999) Stout et al. (1998) Cipollini and Redman (1999) Redman et al. (2001) Karban et al. (2000) Castanera et al. (1996) Cipollini et al. (2002) Cipollini et al. (2002)

Reference

258 C.P. Constabel, R. Barbehenn


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leaves (Melo et al. 2006). Relative growth rates of Helicoverpa zea caterpillars were negatively correlated with tomato leaf and tomato fruit PPO levels (Felton et al. 1989), while Manduca quinquemaculata caterpillars showed greater performance on younger tobacco leaves, which contain higher PPO levels (Kessler and Baldwin 2002). Lymantria dispar caterpillars were unaffected by an eight-fold increase in PPO levels, using mushroom PPO applied to the leaf surface (Barbehenn et al. 2007). Correlations of PPO activity with defense may be confounded by the complexity of PPO gene families. For example, the poplar genome contains as many as 10–12 PPO genes (I.T. Major, L. Tran and C.P. Constabel, unpublished data), but to date, only three PPO genes have been studied. PtdPPO1 is exclusively expressed in damaged leaves, while PtdPPO2 and PtdPPO3 are predominantly expressed in stems, petioles, or roots. Both PtdPPO1 and PtdPPO2 are inducible, but in different tissues (Wang and Constabel 2004). Tissue-specific PPO expression has been most carefully studied in tomato, where seven PPO genes were characterized by Steffens and co-workers (Hunt et al. 1993; Steffens et al. 1994). In elegant work using promoterGUS fusions, in situ hybridization, and immunolocalization, a highly complex cell- and tissue-specific pattern of expression was established (Thipyapong 1997; Thipyapong and Steffens 1997). Each PPO showed a distinctive expression profile, but at least one PPO gene showed constitutive expression for any given tissue. Interestingly, only the PPO-F gene was found to be herbivore-inducible. In addition to systemin and MeJA, pathogen and abiotic stress signals such as salicylic acid and ethylene were also shown to regulate PPO-F, consistent with additional roles of PPO in pathogen or other stress resistance. In general, diverse expression patterns of PPO in tomato and poplar in response to both developmental and stress signals indicate that PPO may have additional stress-related functions in different systems or situations.

12.4 Some Defensive Functions of PPO have been Demonstrated in Transgenic Tomato and Poplar Plants In tomato and poplar, the induction and regulation of PPO in the herbivore defense response, in parallel with many confirmed defense genes, has provided indirect evidence for its role in defense (‘guilt by association’). Nevertheless, it is possible that the herbivore-induced PPO contributes to wound healing and defense against opportunistic pathogens, rather than direct defense against insects. This question can best be addressed using transgenic plants. The availability of PPO cDNAs from a diversity of species has facilitated this approach in species susceptible to genetic transformation. Based on the commercial interest in the role of PPO in browning, several studies reported the successful anti-sense suppression of PPO in potato tuber and apple fruit (Bachem et al. 1994; Murata et al. 2001; Coetzer et al. 2001). Conversely, the overexpression of PPO in transgenic sugarcane resulted in darker juice (Vickers et al. 2005). However such PPO-modified plants were not used to address questions of biological functions or plant defense.

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The defensive roles of PPO were first directly tested with PPO-overexpressing tomato plants, which showed fewer lesions and increased resistance to the bacterial pathogen Pseudomonas syringae pv tomato (Li and Steffens 2002), while antisense PPO-suppressed tomato showed greater susceptibility (more bacterial replication and more lesions; Thipyapong et al. 2004a). These plants also provide the strongest support to date for a defensive role of PPO against insect herbivores. Noctuid caterpillars Heliothis armigera and Spodoptera litura showed negative effects on growth when fed on PPO-overexpressing lines, and conversely, positive effects on growth when fed PPO-suppressed lines (P. Thipyapong, personal communication). In transgenic Populus, overexpression of the induced leaf PPO gene in low PPO poplar lines facilitated assays for roles of PPO against tree-feeding caterpillars. These studies have provided mixed results. First-instar caterpillars of Malacosoma disstria had decreased growth rates on elevated-PPO poplar (Wang and Constabel 2004), but only when experiments were performed in the fall, presumably a result of decreased caterpillar vigor. Similarly, fourth-instar caterpillars of Lymantria dispar had decreased growth rates on elevated-PPO poplar in the winter (Barbehenn et al. 2007). A second species of lymantriid caterpillar, Orgyia leucostigma, had decreased growth rates on elevated-PPO poplars in one experiment, but no negative effects were observed in another experiment (both in the winter). Here it appears that the varying and small effects of large increases in PPO activity in poplar (five to 40-fold increases) would make induced PPO alone ineffective as a defense against these tree-feeding caterpillars. The differences in the apparent effectiveness of overexpressing PPO in tomato vs. poplar may be due to several factors, including the differential susceptibilities of test insects. The cell-specific localization of PPO may also be important; in tomato a significant proportion of overexpressed PPO is found in glandular trichomes (P. Thipyapong, personal communication). This may favor pre-ingestive PPO oxidation of phenolics and avoid the anoxic environment of the gut (see below). Similarly, a rapid oxidation of phenolics in tomato may be favored by the lack of latency for the tomato, but not the poplar enzyme (Constabel et al. 1995, 2000). PPO-overexpressing transgenic poplars have been useful tools for probing other aspects of PPO, such as its stability after ingestion. Defense proteins are predicted to be relatively stable in the harsh conditions found in insect digestive systems, and the recovery of significant amounts of PPO in frass of forest tent caterpillar feeding on transgenic foliage is consistent with this expectation (Wang and Constabel 2004). Furthermore, PPO was activated by its passage through the insect gut, since unlike PPO extracted from leaves, PPO in frass extracts was fully active. Western blot analysis using PPO-specific antibodies showed that PPO in frass migrated at a lower molecular weight, indicating that proteolytic processing at a discrete site occurred in the gut (Wang and Constabel 2004). As mentioned above, PPOs are frequently observed to have a C-terminal proteolytic processing site (Marusek et al. 2006). The biological significance of PPO activation by gut enzymes is not clear, but it may impact its effectiveness.


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12.5 PPO Activity Against Insects: Mechanisms of Action and Limitations The many studies of PPO induction by herbivores attest to the general belief that PPOs play a key role in defense against herbivores. However, since the work of Felton et al. (1989, 1992), studies have rarely examined the mode of action of ingested PPOs. At least three mechanisms have been proposed by which PPO might affect insect herbivores: (1) PPO-generated quinones could alkylate essential amino acids, decreasing plant nutritional quality, (2) redox cycling of quinones may produce oxidative stress in the gut lumen, and (3) phenolic oxidation products, such as quinones and reactive oxygen species (hydrogen peroxide) generated by quinone redox cycling, could be absorbed and have toxic effects on herbivores. The work that has addressed these mechanisms in insect herbivores is summarized below. As originally shown by Felton et al. (1989, 1992), PPO can directly reduce protein quality in vitro, when incubated with dietary protein and chlorogenic acid at ambient oxygen at pH 7.0. The alkylation of essential amino acids with quinones under these conditions significantly decreased noctuid caterpillar performance, and up to 50% of radiolabeled chlorogenic acid was found bound to protein in frass of noctuid caterpillars fed on tomato foliage (Felton et al. 1989). High pH environments, such as the lepidopteran midgut, favor protein alkylation, and several essential amino acids (lysine, histidine, cysteine, methionine) are particularly susceptible to quinone alkylation (Felton et al. 1992). Under optimal conditions for PPO activity, PPO clearly has an effect on protein nutritional quality. However, limiting factors in insect digestive systems may be low oxygen levels and the presence of antioxidants such as ascorbate or glutathione (see below). Another potential effect of PPO is elevated oxidative stress in the gut lumen, which we examined recently using PPO-overexpressing poplar foliage (Barbehenn et al. 2007). High PPO levels had little effect on observed levels of oxidized proteins or semiquinone radical production in two tree-feeding caterpillar species. Coating leaf disks with a commercial PPO (fungal tyrosinase) likewise produced no increase in semiquinone radical levels. These data suggest that there is little increase in quinone formation following ingestion of high levels of PPO in poplar, contrary to expectations based on the model of Felton et al. (1989, 1992) and previous results with forest tent caterpillars (Wang and Constabel 2004). By contrast, Thipyapong and coworkers have recently shown decreased growth rates and decreased nutritional indices of some noctuid caterpillars on PPO-overexpressing tomato lines (P. Thipyapong, personal communication), consistent with post-ingestive mechanism(s) of PPO activity. We are unaware of any work that has examined the potential effect of PPO on oxidative stress or toxicity at the tissue level in insects. The activity of ingested PPO is dependant on the chemical environment of the insect gut, such as oxygen and phenolic substrate levels, reductants, inhibitors, and pH. Surprisingly little work has been done to determine how the physiological conditions present in insect gut fluids influence PPO and other defensive reactions. Phenolic substrates must be present for PPO to be effective, but unfortunately these are typically not analyzed and are assumed to be present at sufficient levels.

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Fig. 12.1 Effect of molecular oxygen concentration on PPO reaction rate [modified from Duckworth and Coleman (1970)]. Reaction mixtures contained PPO (125 ␮g/ml) and pyrocatechol (0.25 mM) in 0.1 M sodium phosphate buffer (pH 7.0)

Reaction rate (arbitrary units)

Likewise, molecular oxygen is an absolute requirement for PPO, and its activity is halted by purging oxygen from the reaction mixture (Duckworth and Coleman 1970; Fig. 12.1). Significantly, the gut contents of caterpillars and grasshoppers contain low steady-state concentrations of oxygen, and are sometimes anaerobic (Johnson and Barbehenn 2000). Oxygen drops from ambient levels of 150 mm Hg (21.0%) to less than 10 mm Hg (1.4%) over a distance of several mm into the foreguts of some caterpillars. The midgut oxygen levels of 0.1–0.5 mm Hg seen in many species would be expected to decrease PPO activities to less than 1% of maximal PPO rates at ambient oxygen levels (Fig. 12.1). Some caterpillar species have enlarged foreguts with much higher oxygen levels than the midgut. Nevertheless, in our work with one such species, Lymantria dispar, we found little evidence for a strong effect of PPO (Barbehenn et al. 2007). The limited oxygen availability in midguts of many insects should be kept in mind when using purified proteins or macerated leaf tissues to model chemical processes within herbivores. Where an impact of PPO on herbivores is found, limiting factors in the gut argue for a preingestive mode of action of PPO. Although pH optima of PPOs are commonly broad, the reactivity of quinones with amino acids in an acidic medium is greatly reduced. Thus, Felton et al. (1992) concluded that PPOs would likely be ineffective against the Colorado potato beetle due to the low pH (5.5–6.5) of the beetle’s midgut. Consistent with this conclusion, no significant decreases in the levels of four essential amino acids were found in the feces of L. decemlineata that fed on potato leaves from varieties with higher PPO levels (Castanera et al. 1996). Grasshopper gut pH is also acidic, but we are unaware of work on the effects of PPO in the Orthoptera. The high pH found in lepidopteran midguts (ca. pH 9–10) would be expected to decrease the activities of ingested PPO, but the basic conditions favor protein alkylation (Felton et al. 1989). Extensive work by food scientists has identified many PPO inhibitors that reduce browning of processed foods. Among these inhibitors, ascorbate is ubiquitous in leaves and present at high levels, and would be co-ingested with PPO. When present in midgut fluid or in vitro reaction mixtures at 0.2–0.5 mM, ascorbate can chemically reduce quinones and semiquinone radicals, thereby limiting the effectiveness of PPO as an oxidative defense (Martinez-Cayuela et al. 1988; Janovitz-Klapp et al. 1990; Felton and Duffey 1992; Barbehenn et al. 2007). Levels of ascorbate

1.0 0.8 0.6 0.4 0.2 0.0 0

25

50

75

100

[O2] (mm Hg)

125

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in the midgut fluids of tree-feeding caterpillars feeding on young trembling aspen, red oak, and hybrid poplar foliage are sufficiently high to prevent the net production of quinones by PPO (Barbehenn et al. 2003, 2007). Furthermore, an ascorbate recycling system is considered to be central to the antioxidant defenses of tree-feeding caterpillars (Barbehenn et al. 2001). However, it is not known whether the ascorbate ingested by tomato-feeding caterpillars, such as noctuids, is maintained in their gut fluids at sufficiently high levels to inhibit PPO. Thiols such as glutathione and cysteine also decrease the net production of quinones by PPO and may inhibit the enzyme directly (Negishi and Ozawa 2000). In leaves, glutathione concentrations are roughly 5% as high as ascorbate levels, but can be equal to ascorbate levels in caterpillar midgut fluids (Barbehenn et al. 2001, 2003). Midgut glutathione at 50–100 ␮M in tree-feeding caterpillars is thus theoretically high enough to inhibit PPO activity (Barbehenn et al. 2003; Nagai and Suzuki 2003; Aydemir and Akkanh 2006), although apparently higher levels of cysteine are necessary to inhibit PPO from some plants (e.g., JanovitzKlapp et al. 1990). Other potential PPO inhibitors reported include simple phenolics and quercetin (Walker and McCallion 1980; Le Bourvellec et al. 2004; Nerya et al. 2004).

12.6 PPO and Pathogen Defense A role for PPO in defense against pathogens has been postulated from the earliest days of PPO research. This hypothesis has been supported by many correlative studies, such as the upregulation of PPO in pathogen-challenged plants (reviewed in Constabel et al. 1996; Mayer 2006). Pathogen-induced PPO activity continues to be reported for a variety of plant taxa, including monocots and dicots (e.g., Chen et al. 2000; Deborah et al. 2001). Similarly, studies describing correlations of high PPO levels in cultivars or lines with high pathogen resistance continue to provide support for a pathogen defense role of PPO (Raj et al. 2006). Several groups have also attempted to correlate the protective effects of rhizosphere bacteria with an induction of defense enzymes including PPO, with mixed success (Chen et al. 2000; Ramamoorthy et al. 2002). Direct evidence for a role of PPO in inhibiting pathogen ingress or growth comes from transgenic tomato plants with enhanced or suppressed PPO levels. When challenged by the bacterial pathogen Pseudomonas syringae pv tomato, PPO-overexpressing plants showed reduced bacterial growth, whereas PPO antisense-suppressed lines supported greater bacterial numbers (Li and Steffens 2002; Thipyapong et al. 2004a). These studies are the only direct demonstrations to date of PPO’s importance in pathogen defense. Whether such a function extends to other types of pathogens such as fungi remains to be tested. In poplar, infection with Melampsora medusae (a foliar rust pathogen) does not induce PPO, but represses its expression together with many other herbivore defense genes (Miranda et al. 2007).

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The demonstration that PPO can inhibit bacterial plant pathogens suggests that, in addition to effects on some herbivores, PPO is important for inhibiting microbes introduced into damaged leaves via the mouthparts of feeding insects. These might be opportunistic bacteria or pathogens that are vectored by specific insects, although little is known about the microflora of caterpillar mouthparts and digestive systems. Thus, the distinction between pest and pathogen defenses may be artificial, and these may be seen as synergistic and complementary responses. This contrasts with the view that pest and pathogen defense responses are mutually exclusive. This hypothesis was based on early work on tomato showing that salicylic acid, a potent inducer of pathogen defense and systemic acquired resistance, inhibits the jasmonate-regulated herbivore defense response (Doares et al. 1995; Thaler et al. 2002). Current models of defense signaling outline a complex and overlapping set of responses, regulated by jasmonate, ethylene, and salicylic acid-based signals, leading to several possible outcomes (Devoto and Turner 2005). Functionally, crosstalk is supported by the observation of herbivore-induced resistance of Arabidopsis against microbial pathogens, including P. syringae (De Vos et al. 2006). In tomato, the PPO-F gene is induced during both the wound response and infection with the pathogen P. syringae (Thipyapong 1997, 2004a). To date, no clear mechanism for the potential anti-pathogen effects of PPO has been demonstrated. Li and Steffens (2002) suggest several possibilities, including (1) general toxicity of PPO-generated quinones to pathogens and plant cells, accelerating cell death, (2) alkylation and reduced bioavailability of cellular proteins to the pathogen, (3) cross-linking of quinones with protein or other phenolics, forming a physical barrier to pathogens in the cell wall, and (4) quinone redox cycling leading to H2 O2 and other reactive oxygen species (Jiang and Miles 1993). While reactive oxygen species are known to be important factors in plant pathogen interactions and defense signaling, and PPO is implicated in the formation of melanin-like polymers in potato blackspot lesions (Stevens et al. 1998), none of these hypotheses of how PPO might affect pathogens has been tested rigorously so far.

12.7 Conclusions and Future Directions Although the transgenic approach has led to the direct demonstration of the efficacy of inducible PPO as a defense against some lepidopteran herbivores in some cases, the mechanisms of action against these attackers are still unclear. More detailed analyses of midgut chemistry in a greater variety of insects feeding on high-PPO foliage will begin to address this question. In particular, the extent of quinone binding to protein and amino acids in the gut contents of herbivores needs to be established. The interpretation of feeding studies would be enhanced by testing not only PPO levels and insect performance, but consumption rate and plant nutritional and chemical quality, so that deterrence or compensatory feeding can be detected. In addition, our knowledge of PPO effects would benefit from greater attention to endogenous PPO substrates, rather than simply extrapolating from substrate speci-


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ficities observed in vitro. In few cases have the probable substrates been identified, yet the biochemical reactivities of PPO-produced quinones are dependent on the specific structure (Jiang and Miles 1993). Much recent work on PPO has emphasized potential roles in defense. Nevertheless, alternate roles for this enzyme are likely and have been demonstrated. For example, antisense PPO tomato plants have enhanced pathogen resistance and drought tolerance (Thipyapong et al. 2004b), and PPO-like enzymes can act as hydroxylases in secondary metabolism (Steiner et al. 1999; Nakayama et al. 2000; Cho et al. 2003). Given the tremendous variation in PPO expression patterns, activity levels, and potential substrates in different species, similar variation in the adaptive roles played by PPO in defense and other processes may be anticipated. Acknowledgments The authors would like to thank Piyada Thipyapong for providing unpublished data. Grant support for work in the authors’ laboratories is from the Natural Sciences and Engineering Research Council (NSERC) of Canada (CPC), the University of Victoria’s Centre for Forest Biology (CPC), and the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2004-35302-14840 to RVB and CPC.

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Chapter 13

Action of Plant Defensive Enzymes in the Insect Midgut Hui Chen, Eliana Gonzales-Vigil and Gregg A. Howe

Insect feeding activates the expression of host plant defensive proteins that exert direct effects on the attacker. In addition to the well-studied proteinase inhibitors, the plant’s defensive protein arsenal includes enzymes that disrupt various aspects of insect digestive physiology. In this chapter, we summarize recent studies on isoforms of arginase and threonine deaminase (TD) that degrade the essential amino acids arginine and threonine, respectively, in the alkaline environment of the lepidopteran gut. We also discuss a vegetative storage protein (VSP2) whose phosphatase activity is responsible for potent insecticidal effects on dipteran and coleopteran insects. A common feature of VSP2, TD, and arginase is their wound-inducible expression via the jasmonate signaling pathway. Jasmonate-regulated defensive enzymes may have evolved from pre-existing housekeeping enzymes that catabolize essential nutrients during normal plant development. The application of proteomics-based approaches to identify plant proteins in the digestive tract of phytophagous insects is facilitating the discovery of novel plant proteins with insecticidal activity.

13.1 Introduction The optimal growth of insect herbivores depends on their ability to acquire amino acids from dietary protein. Like other animals, leaf-eating insects require the essential amino acids arginine, threonine, isoleucine, leucine, lysine, histidine, methionine, phenylalanine, tryptophan, and valine (Chang 2004). The low protein content of plant tissue poses a major challenge to insects that require these nutrients, as protein is both the major macronutrient and the most commonly limiting nutrient for insect growth (Mattson 1980; Bernays and Chapman 1994). A large body of evidence indicates that many plant defensive compounds impair herbivore performance by restricting the availability of amino acids or other plant-derived nutrients. Much of this literature concerns plant secondary metabolites such as tannins and phenolic

G.A. Howe DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA e-mail: [email protected]


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resins that interfere with the digestibility of dietary protein (Feeny 1976; Rhoades and Cates 1976; Berenbaum 1995). Higher plants also produce a variety of proteins that disrupt amino acid acquisition and other aspects of insect digestive physiology (Felton 1996). Proteinase inhibitors (PIs) initially described by Ryan and colleagues are perhaps the best example of this type of post-ingestive defense (Ryan 1990; Jongsma and Beekwilder this volume). Upon consumption of plant tissue by the herbivore, PIs bind to and inhibit digestive proteases in the gut lumen. The negative effect of PIs on herbivore growth results from a compensatory response by the insect to overproduce digestive proteinases, which, in turn, depletes pools of essential amino acids (Broadway and Duffey 1986). Because PIs are not catalytic, their ability to thwart insect growth is dependent on accumulation to relatively high concentrations in the digestive system. Plant enzymes have the potential to exert defensive effects at much lower concentrations. This idea has received relatively little attention until recently (Duffey and Stout 1996; Felton 1996; Chen et al. 2005; Felton 2005). Research on plant enzymes that serve a post-ingestive role in defense has focused mainly on polyphenol oxidase and other oxidative enzymes that covalently modify dietary protein and thus impair the digestibility of plant food (Constabel et al. 1995; Duffey and Stout 1996; Felton 1996; Wang and Constabel 2004; Constabel and Barbehenn this volume). Other defensive enzymes, including cysteine proteases, target structural components of the insect digestive apparatus (Pechan et al. 2002; Konno et al. 2004; Mohan et al. 2006). The collective evidence thus indicates that plant enzymes play a pivotal role in anti-insect defense, and broadens the view that secondary metabolites are the major determinant of host plant selection by insects (Fraenkel 1959; Berenbaum 1995). Plant proteins that serve a post-ingestive role in defense are typically synthesized in response to wounding and herbivore attack. This central tenet of induced resistance was established 35 years ago by Green and Ryan’s pioneering work on serine PIs in potato and tomato (Green and Ryan 1972). Induced expression of PIs and other defense-related proteins is controlled in large part by the jasmonate signaling pathway (Walling 2000; Kessler and Baldwin 2002; Gfeller and Farmer 2004; Howe 2004; Schilmiller and Howe 2005). Recent research on jasmonate-signaled processes indicates that enzymes previously thought to be involved in primary metabolism serve an important role in post-ingestive defense. In this chapter, we summarize recent findings on two jasmonate-regulated enzymes, arginase and threonine deaminase (TD), which have the capacity to degrade essential amino acids in the lepidopteran gut. Recent research concerning the role of jasmonate-regulated vegetative storage proteins (VSPs) in plant defense against insect herbivores is also discussed.

13.2 Arginase L-arginine (Arg) is one of the most functionally diverse amino acids in living cells. In addition to being a building block for proteins, Arg is a precursor for the biosynthesis of polyamines, agmatine, and proline, as well as the cell-signaling molecules glutamate, ␥-aminobutyric acid, and nitric oxide (NO; Fig. 13.1). Two of the most

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Fig. 13.1 Arginine metabolism in higher plants. Arginine is a substrate for both arginase (ARG) and nitric oxide synthase (NOS). NOS catalyzes the synthesis of nitric oxide (NO) by a twostep reaction involving an N-hydroxy-arginine (NOHA) intermediate. Metabolism of arginine via the arginase-ornithine decarboxylase (ODC) pathway gives rise to proline, glutamate, and simple polyamines. Arginine is also metabolized by arginine decarboxylase to agmatine (not shown), which also is used as a precursor for polyamine synthesis

intensively studied pathways of Arg metabolism are those involving arginase and NO synthase (NOS). Arginase (L-arginine amidino hydrolase, EC 3.5.3.1) is a binuclear manganese metalloenzyme that hydrolyzes Arg to urea and ornithine, the latter of which is a precursor for polyamine biosynthesis. In animal systems, increased arginase expression stimulates the production of polyamines that promote cell proliferation and wound healing (Satriano 2004). In contrast to the growth-promoting effects of polyamines are the cytostatic effects of NO produced by activated macrophages. The switch between the arginase and NOS branches of Arg metabolism is controlled by various signaling and metabolic pathways that regulate Arg availability (Mori and Gotoh 2004; Satriano 2004). Because arginase and NOS compete for a common substrate (Fig. 13.1), increased arginase expression can effectively inhibit the NOS pathway. Many animal pathogens, for example, induce arginase expression as a means of evading NO-mediated host defenses (Vincendeau et al. 2003). Very little is known about the physiological function of plant arginase. A prerequisite for addressing this question is the unambiguous identification of plant genes that encode arginase. Because the predicted sequences of plant arginase are more similar to agmatinase than to authentic arginases from vertebrates, fungi, and bacteria, some workers have suggested that plant genes annotated as arginase may encode agmatinase or a related amidinohydrolase (Perozich et al. 1998; Sekowska et al. 2000). Recent work on arginases from cultivated tomato (Solanum lycopersicum) helped to resolve this issue. The tomato genome contains two arginase genes designated ARG1 and ARG2 (Chen et al. 2004). Both genes are predicted to encode 338-amino-acid proteins, which are 89% identical to each other. Heterologous expression studies showed that both enzymes have very similar biochemical properties, including high specificity for Arg. The apparent K m of Arg is ∼30 mM for both isozymes, which is consistent with K m values reported for arginases purified from various plant tissues. These studies demonstrate that tomato ARG1 and 2, despite their sequence similarity to agmatinase and phylogenetic distinction from non-plant arginases, are genuine arginases (Chen et al. 2004). Additional insight into the function of plant arginase was obtained by analysis of the enzyme’s tissue-specific expression pattern. Tomato ARG1 and ARG2

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genes, for example, are expressed to their highest levels in reproductive tissues (Chen et al. 2004). This observation agrees with previous studies showing that tomato ovaries and immature fruit contain relatively high levels of arginase activity (Alabadi et al. 1996). Research on soybean indicates that arginase may be involved in mobilizing Arg during early seedling germination (Goldraij and Polacco 1999, 2000). The first clue that arginase may have a role in plant defense came from the observation that expression of tomato ARG2 (but not ARG1) is massively induced in foliar tissue in response to mechanical wounding and methyl jasmonate treatment (Chen et al. 2004). Induced expression of ARG2 was accompanied by increases in arginase activity, and was abolished in the jasmonic acid insensitive1 (jai1) mutant that is defective in the jasmonate signaling pathway (Li et al. 2004). The absence of detectable ARG2 expression in jai1 plants suggests that this isoform is not essential for normal plant growth and development. An unusual feature of plant arginase is its ability to metabolize Arg at high pH. The pH optimum of tomato ARG2 is ∼9.5, with little or no activity detected at pH 7.0 (Fig. 13.2). The dependency of ARG2 activity on high pH led to the idea that the enzyme, following its wound-induced accumulation in tomato leaves, acts in the alkaline midgut of lepidopteran insects that feed on tomato foliage; by depleting the pool of Arg available for uptake into the intestine, arginase may reduce the nutritional quality of the plant food. This hypothesis is consistent with the proposal by Broadway and Duffey (1988) that reduced availability of basic amino acids (i.e., Arg and lysine) increases the toxicity of PIs. Several lines of evidence support a role for arginase in post-ingestive defense. First, jai1 mutant plants that fail to express ARG2 are compromised in defense against herbivore attack (Li et al. 2004; Chen et al. 2005). Second, the midgut content of tobacco hornworm (Manduca sexta) larvae reared on foliage from induced tomato plants contained high levels of ARG2 activity (Chen et al. 2005). Finally, increased arginase activity in midgut extracts was associated with reduced levels of free Arg in the same extract (Chen

Fig. 13.2 pH optimum of tomato arginase2. A hexahistidine-tagged derivative of tomato arginase2 was expressed in E. coli and purified by nickel-affinity chromatography (Chen et al. 2004). The recombinant enzyme was assayed for arginase activity in buffer systems (K-PO4 , triangles; Tris, diamonds; glycine, squares; K-PO4 , circles) adjusted to the indicated pH

Arginase activity (arbitrary units)

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et al. 2005). These results indicate that JA-regulated ARG2 in tomato leaves is active in the M. sexta digestive tract, where it catabolizes Arg. A transgenic approach was used to determine whether increased expression of foliar arginase is sufficient to reduce caterpillar performance (Chen et al. 2005). Transgenic tomato plants that overexpress the ARG2 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter were generated by Agrobacteriummediated transformation. The constitutive level of arginase activity in unwounded leaves of selected 35S::ARG2 lines far exceeded that in herbivore-damaged wildtype leaves. High arginase activity in these plants did not result in obvious morphological or reproductive phenotypes, nor did it significantly alter the level of free Arg in 35S::ARG2 leaves (Chen and Howe unpublished data). Feeding trials conducted with two independent 35S::ARG2 lines showed that the average weight of larvae grown on transgenic plants was significantly less than that of larvae reared on wild-type plants. These bioassays also showed that larvae consumed more foliage from wild-type plants than from 35S::ARG2 plants. Arginase activity in midgut extracts from 35S::ARG2-reared larvae was significantly greater than that in wildtype-reared larvae, and this activity was associated with reduced levels of midgut Arg. Thus, ingestion of foliar arginase by M. sexta larvae results in the depletion of midgut Arg and reduced larval growth (Chen et al. 2005). To test the idea that reduced growth of M. sexta caterpillars on 35S::ARG2 plants is a direct consequence of Arg deficiency in the diet, feeding trials were performed with plants that were sprayed with a solution of 10 mM Arg (Fig. 13.3). This treatment did not affect larval performance on wild-type plants, suggesting that free Arg is not a limiting factor for insect growth on this host. However, significant improvement of larval growth was observed on Arg-supplemented 35S::ARG2 plants

larval weight (g)

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+

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Fig. 13.3 Exogenous arginine partially rescues the reduced growth of M. sexta larvae reared on transgenic 35S::ARG2 plants. Plants of the indicated genotype were sprayed with 10 mM arginine (+) or a mock control (−) prior to challenge with newly hatched larvae. Larval weights were determined 10 days after the start of the feeding trial. Data show the mean and SD of at least 23 larvae per host genotype. Lower case letters denote significant differences at pSer/Thr) specific lectin isolated from the seeds of Salvia sclarea. J Biomed Sci 12:167–184 Wu J, Luo X, Guo H, Xiao J, Tian Y (2006) Transgenic cotton, expressing Amaranthus caudatus agglutinin, confers enhanced resistance to aphids. Plant Breed 125:390–394 Yagi F, Hidaka M, Minami Y, Tadera K (1996) A lectin from leaves of Neoregelia flandria recognizes D-glucose, D-mannose and N-acetylglucosamine, differing from the mannose-specific lectins from other monocotyledonous species. Plant Cell Physiol 37:1007–1012 Yao J, Pang Y, Qi H, Wan B, Zhao X, Kong W, Sun X, Tang K (2003) Transgenic tobacco expressing Pinellia ternata agglutinin confers enhanced resistance to aphids. Transgenic Res 12:715–722 Young NM, Oomen RP (1992) Analysis of sequence variation among legume lectins. A ring of hypervariable residues forms the perimeter of the carbohydrate-binding site. J Mol Biol 228:924–934 Zhang W, Peumans WJ, Barre A, Houles-Astoul C, Rovira P, Rougé P, Proost P, Truffa-Bachi P, Jalali AAH, Van Damme EJM (2000) Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants. Planta 210:970–978 Zhou X, Li XD, Yuan JZ, Tang ZH, Liu WY (2000) Toxicity of cinnamomin – a new type II ribosome-inactivating protein to bollworm and mosquito. Insect Biochem Mol Biol 30:259–264 Zhu K, Huesing JE, Shade RE, Bressan RA, Hasegawa PM, Murdock LL (1996) An insecticidal N-acetylglucosamine-specific lectin gene from Griffonia simplicifolia (Leguminosae). Plant Physiol 110:195–202 Zhu-Salzman K, Salzman RA (2001) Functional mechanics of the plant defensive Griffonia simplicifolia lectin II: resistance to proteolysis is independent of glycoconjugate binding in the insect gut. J Econ Entomol 94:1280–1284 Zhu-Salzman K, Salzman RA, Koiwa H, Murdock LL, Bressan RA, Hasegawa PM (1998) Ethylene negatively regulates local expression of plant defense lectin genes. Physiol Plant 104:365–372 Zhu-Salzman K, Shade RE, Koiwa H, Salzman RA, Narasimhan M, Bressam RA, Hasegawa PM, Murdock LL (1996) Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II. Proc Natl Acad Sci USA 95:15123–15128

Section III

Defense Signaling

Part A

Activation of Plant Defenses

Chapter 15

Systemins and AtPeps: Defense-Related Peptide Signals Javier Narváez-Vásquez and Martha L. Orozco-Cárdenas

Plants have evolved different families of functionally related peptide signals (15–23 amino acids) that serve to amplify the defense response against insect/herbivore and pathogen attacks through the activation of the octadecanoid signaling pathway. The peptides are derived from larger precursor proteins and released into the extracellular space to interact with membrane receptors by still unknown processing mechanisms. Besides scarce sequence similarities among defense-related peptide signals, their functional properties still suggest some common ancestral origin for defense peptide signaling systems in plants and animals.

15.1 Introduction In the Solanaceae, a family of defense-related peptide hormones called systemins are involved in the activation of defense genes in response to wounding by herbivore attacks (Ryan and Pearce 2003). Tomato systemin (Sys) is an 18-amino acid oligopeptide located at the C-terminus of a cytosolic precursor protein of 200 amino acids called prosystemin (ProSys; Fig. 15.1A; Ryan and Pearce 1998). Upon wounding, Sys seems to be proteolytically processed from ProSys and released into the cell wall apoplast, where Sys interacts with a membrane receptor to activate defense signaling. In addition to Sys, tomato plants possess three functionally related hydroxyproline-rich glycopeptides (called TomHypSys I, II, and III) of 15–20 residues that are synthesized from a single polyprotein precursor unrelated to ProSys (Pearce and Ryan 2003; Fig. 15.1C). The ProHypSys precursor proteins are synthesized with a signal peptide through the secretory pathway and localized in the cell wall (Narváez-Vásquez et al. 2005). Recently, a novel wound-inducible, defense-related peptide signal has been identified in Arabidopsis (Huffaker et al. 2006a). The Arabidopsis peptide1 (AtPep1; Fig. 15.1D) is part of a small family of peptide signals involved in the innate immune J. Narváez-Vásquez Department of Botany and Plant Sciences, University of California Riverside, Riverside, CA 92521, USA e-mail: [email protected]


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A 1 66 131 197

MGTPSYDIKNKGDDMQEEPKVKLHHEKGGDEKEKIIEKETPSQDINNKDTISSYVLRDDTQEIPK MEHEEGGYVKEKIVEKETISQYIIKIEGDDDAQEKLKVEYEEEEYEKEKIVEKETPSQDINNKGD DAQEKPKVEHEEGDDKETPSQDIIKMEGEGALEITKVVCEKIIVREDLAVQSKPPSKRDPPKMQTD NNKL

B 1 MRVLFLIYLILSPFGAEARTLLENHEGLNVGSGYGRGANLPPPSPASSPPSKEVSNSVSPTRTDE 66 KTSENTELVMTTIAQGENINQLFSFPTSADNYYQLASFKKLFISYLLPVSYVWNLIGSSSFDHDL 131 VDIFDSKSDERYWNRKPLSPPSPKPADGQRPLHSY C 1 MISFFRAFFLIIIISFLIFVGAQARTLLGNYHDDEMLIELKLESGNYGRTPYKTPPPPTSSSPTHQ 67 EIVNGRHDSVLPPPSPKTDPIIGQLTTITTTPHHDDTVAAPPVGGRHDYVASPPPPKPQDEQRQII 133 ITSSSSTLPLQASY D 1 MEKSDRRSEESHLWIPLQCLDQTLRAILKCLGLFHQDSPTTSSPGTSKQPKEEKEDVTME 61 KEEVVVTSRATKVKAKQRGKEKVSSGRPGQHN

Fig. 15.1 Deduced amino acid sequences of the precursors of defense-related peptides in plants. (A) Tomato systemin precursor protein, prosystemin (200 residues). The systemin sequence (18 residues) at the C-terminus is bold and underlined. Five imperfect repeats (underlined) suggest that the ProSys gene has resulted from gene duplication and elongation events (see text for discussion). (B–C) Tobacco (B, 165 residues) and tomato (C, 146 residues) hydroxyproline-rich systemin polyprotein precursors. Sequences corresponding to the bioactive peptides (15–20 residues) are bold and underlined. The putative signal peptide sequence in both precursors is shown in italics. A short conserved sequence at the N-terminus is also underlined. (D) Arabidopsis peptide1 (AtPep1) precursor protein (92 residues). The sequence of AtPep1 (23 residues) is bold and underlined

response against pathogens, with orthologs in species from several plant families, including the Solanaceae. Like Tomato Sys, AtPep1-related peptides are synthesized from larger precursor proteins that lack a classical signal sequence (Huffaker et al. 2006). The activation of defense genes by systemins and AtPep1, including the genes encoding their own precursor proteins, is mediated by the octadecanoid signaling pathway (Ryan and Pearce 2003; Huffaker et al. 2006). In this pathway (Schaller et al. 2005), linolenic acid is released from cell membranes by the action of phospholipases, and transformed by a series of enzymatic reactions in the oxylipins phytodienoic acid (OPDA) and jasmonic acid (JA). These oxylipins are potent inducers of defense gene activation (see other contributions in this volume). Recent genetic evidence and localization studies suggest that JA, or JA-derived oxylipins function as the systemic signals involved in the systemic wound response against herbivores in tomato (Howe 2005; Wasternack et al. 2006), and in the pathogen-induced systemic acquired resistance (SAR) in Arabidopsis plants (Truman et al. 2007). The cumulative evidence thus indicates that plants have evolved a peptide-based signaling system that activates and amplifies the synthesis of widely spread oxilipin signals in plants for defense against pathogen and herbivore attacks. This

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is analogous to the activation of the innate immune and inflammatory defense responses mediated by polypeptide cytokines in animals through the production of eicosanoid lipid signals (Bergey et al. 1996), and suggest that defense-related peptide signals may have evolved from a similar ancestor gene in eukaryotes. The genetic, biochemical, and functional properties, and the evolution of the aforementioned plant defense peptide signals are further discussed in this chapter.

15.2 Tomato Systemin (Sys) Sys was the first extracellular signal peptide discovered in plants (Pearce et al. 1991), and it is one of the best studied (Ryan and Pearce 1998). Sys is involved in the activation of more than 20 wound-inducible genes in some species of the Solanaceae family, and shares several characteristics with peptide hormones in animals and yeast (Harris 1989). First, Sys is synthesized as a larger precursor protein, suggesting that it should be released by proteolytic cleavage (Fig. 15.1A; McGurl et al. 1992). Second, Sys is active at very low concentrations. In a plant bioassay, femtomol amounts of Sys supplied to young excised tomato plants through their cut stems cause the leaves to accumulate defense proteinase inhibitor proteins (PIs) within 24 hours (Pearce et al. 1991). In addition, subnanomolar concentrations of Sys cause a rapid increase in the pH of the extracellular medium of cell suspension cultures of Lycopersicon peruvianum, known as the alkalinization response (Felix and Boller 1995). Third, Sys action is mediated by its interaction with a membrane receptor at the cell surface, indicating that Sys should be released into the extracellular space to exert its biological activity (Scheer and Ryan 1999, 2002).

15.2.1 The ProSys Gene and Protein The tomato genome possesses a single copy of the ProSys gene. In addition to Sys located at the C-terminus, there are five imperfect repeats of unknown function in ProSys, indicating that the ProSys gene resulted from several cycles of gene duplication and elongation events (McGurl and Ryan 1992). ProSys homologs have only been found in species of the Solaneae subtribe of the Solanaceae family, including tomato, potato, bell pepper, and nightshade, but it is not found in tobacco or Arabidopsis (Constabel et al. 1998). Pairwise analysis of the deduced amino acid sequences of ProSys orthologs indicate that there is a high degree of conservation among them (73%–88% identity). Synthetic Sys from all investigated Solaneae species are capable of inducing defense PIs in the tomato plant bioassay (Constabel et al. 1998). The ProSys gene is under tissue/cell-type specific, developmental, and environmental regulation (Ryan and Pearce 1998). Low constitutive levels of ProSys mRNA are found within the vascular phloem parenchyma (PP) cells of leaves,

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petioles, stems, but not in roots (Narváez-Vásquez and Ryan 2004). ProSys is also developmentally regulated in all floral organs, where high levels of ProSys mRNA and protein accumulate. In addition, ProSys gene expression is also upregulated in aerial vegetative organs by mechanical wounding, chewing insects, JA and its methyl ester (MeJA), plant and pathogen-derived oligosaccharides, and other pathogen elicitors (Ryan and Pearce 1998). ProSys does not comprise a predicted signal sequence, transmembrane domains, or ER/Golgi-dependent post-translational modifications, suggesting that the Sys precursor is synthesized on free ribosomes in the cytoplasm (McGurl et al. 1992). The amino acid composition of ProSys (Fig. 15.1A) is characterized by an abundance of acidic amino acids (27.5%), but also a large number of basic Lys residues (15%). ProSys contain stretches of alternating Lys (K) and Glu (E) residues, resembling the so-called ‘KEKE’ motifs (Realini et al. 1994). Similar motifs have been shown to be involved in protein–protein interactions through the formation of ‘polar zippers’ (Zhang et al. 1997).

15.2.2 ProSys Processing Sys is an internal peptide that should be released from its protein precursor by proteolytic cleavage by a processing peptidase, similar to other peptide hormones in eukaryotes (Seidah and Chretien 1997). The processing sites and the responsible enzyme(s) that cleave(s) Sys from ProSys are currently unknown. Unlike most prohormone precursors in animals and yeast, ProSys is not synthesized through the secretory pathway. ProSys does not have classical dibasic sites for processing by subtilisin/kexinlike, prohormone convertases (PCs; Seidah and Chretien 1997), except for a dibasic KR sequence present in the middle of Sys (Fig. 15.1A and Table 15.1). A Sysbinding protease in tomato cell membranes related to furin in the PC family may cleave Sys at this site (Schaller and Ryan 1994). The protease may be involved in Sys turnover, since the proteolytic products have significantly reduced biological activities (Pearce et al. 1993). Accordingly, substitution of these basic residues with chemically-modified amino acids increases the half-life and activity of the modified Sys (Schaller 1998). ProSys is also enriched in acidic amino acid doublets (E/D-E/D, Fig. 15.1). In fact, the Sys sequence is flanked by Asp residues at both the N- and the C-termini. Interestingly, acidic pairs of amino acids are also present in the protein precursors of Arabidopsis AtPep peptides (Fig. 15.1D; Huffaker et al. 2006). Plant endoproteases with specificities for P1 Asp residues have been reported (D’Hont et al. 1993; Francois et al. 2002; Rojo et al. 2004; Coffeen and Wolpert 2004), and several peptidases are known to be wound-inducible (Pautot et al. 1993; Bergey et al. 1996; Schaller and Ryan 1996; Horn et al. 2005). Notably, the processing enzyme of the signal-less precursor protein of the potent inflammatory cytokine interleukin-1␤ (IL-1␤) in animal cells is a caspase-like Cys protease that requires substrates with Asp adjacent


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Table 15.1 Sequence comparison and posttranslational modifications of defense-related peptides in plants. Proline (P), hydroxyproline (O), threonine (T), and serine (S) residues are bold Peptide1

Sequence

Pentose units

Sys TomHypSys I TomHypSys II TomHypSys III TobHypSys I TobHypSys II AtPep1

AVQSKPPSKRDPPKMQTD RTPYKTOOOOTSSSPTHQ GRHDYVASOOOOKPQDEQRQ GRHDSVLOOOSOKTD RGANLPOOSOASSOOSKE NRKPLSOOSOKPADGQRP ATKVKAKQRGKEKVSSGRPGQHN

0 8–17 12–16 10 9 6 0

1

Sys, tomato systemin; TomHypSys, tomato hydroxyproline-rich glycopeptides I, II and III; TobHypSys, tobacco hydroxyproline-rich glycopeptides I and II, AtPep1, Arabidopsis peptide1

and N-terminal to the scissile peptide bond (P1 residue; Howard et al. 1991). Thus, it is tempting to speculate that the ProSys precursor is processed by a protease that cleaves at the COOH side of Asp (P1 ), releasing N-terminal extended Sys peptides that could be trimmed by aminopeptidases in the cytosol (Walling 2006). The possibility that ProSys is secreted into the apoplast and subsequently processed by extracellular proteases was investigated (Dombrowski et al. 1999). Recombinant ProSys was incubated with apoplastic wash fluids of tomato leaves and the culture medium of L. peruvianum cells. At different times the integrity of ProSys was assessed by SDS-PAGE and protein blotting using anti-Sys antibodies. There was no evidence for ProSys processing enzymes in the apoplast or media (Dombrowski et al. 1999). Evidence that ProSys is synthesized and processed within PP cells comes from direct immunocytochemical studies, using affinity-purified antibodies raised against full-length ProSys, Sys, and ⌬ ProSys (ProSys lacking the Sys sequence; NarváezVásquez and Ryan 2004). The studies indicated that ProSys is localized in the cytosol and the nucleus of PP cells in leaves, petioles, stems, and floral organs of tomato and potato. The amount of labeling increased significantly in leaves upon wounding or MeJA treatment of tomato plants (Fig. 15.2A). The nucleo-cytoplasmic localization pattern was also observed in all cell types of leaf sections from transgenic tomato plants overexpressing the ProSys gene (Narváez-Vásquez and Ryan 2004). Indirect evidence for intracellular proteolytic processing of ProSys, comes from a recent study in which a chimeric gene encoding a modified ProSys protein, containing a decapeptide sequence of the mosquito trypsin-modulating oostatic factor (TMOF) in the place of Sys, was expressed in transgenic tobacco plants (Tortiglione et al. 2003). The ProSys-TMOF chimeric gene was transcribed and translated into the modified protein within transgenic tissue, and the TMOF decapeptide was detected by HPLC electrospray mass spectrometry (HPLC/ESI-MS) in extracts from transgenic tobacco tissue and protoplasts, but not in extracts from wild type control plants. Only traces of TMOF peptide were detected in the incubating media of the protoplasts, indicating that most if not all of the foreign peptide was being compartmentalized within the cell protoplasts, and was not secreted to the external medium (Tortiglione et al. 2003). Even though tobacco does not have a ProSys gene,

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(A)


(B)

Fig. 15.2 Immuno-cytochemical localization of ProSys and ProHypSys in vascular bundles of tomato leaves. Leaf samples from two-week-old tomato plants exposed for 18 hours to methyl jasmonate vapors were fixed and immuno-gold labeled as previously described (Narváez-Vásquez and Ryan 2004). (A) Silver-enhanced, immuno-gold labeled ProSys localized in vascular phloem parenchyma (PP) cells visualized under the confocal microscope. (B) Transmission electron micrograph showing immuno-gold labeled ProHypSys in the cell walls of PP cells (Narváez-Vásquez et al. 2005). Arrows point to gold particles. pp, phloem parenchyma; cc, companion cell; se, sieve elements; x, xylem vessel. Bars = A, 5.0 ␮m; B, 0.5 ␮m

this result suggests that ProSys is synthesized, processed and/or stored intracellularly in tobacco leaves, most probably in a similar fashion as in tomato. Genetic screens have been conducted in tomato to find mutants in the Sys signaling pathway (Howe and Ryan 1999). These efforts have led to the identification of several mutants incapable to respond to Sys, wounding, or JA (Howe 2005). However, mutations that affect the processing of ProSys have not been identified yet. The failure of these screens to identify genes required for processing, compartmentalization and/or transport of ProSys or Sys suggests that the ProSys processing pathway involves genes that are either redundant or required for cell/plant viability.

15.2.3 The Sys Signaling Pathway A Sys receptor protein, SR160, has been isolated and its gene cloned (Scheer and Ryan 1999, 2002). SR-160 belongs to a family of Ser/Thr receptor kinases with extracellular Leu-rich repeats, which was found to be highly similar to the Arabidopsis brassinolide receptor BRI1 (Scheer and Ryan 2002). Brassinolides are


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a class of plant steroid hormones with different roles in plant development, but not in plant defense. Isolation of the tomato brassinolide receptor confirmed that it is also the Sys receptor (Montoya et al. 2002). The function of SR160 as the Sys receptor was reconfirmed by expressing SR160 cDNA in tobacco plants (Scheer et al. 2003). Tobacco plants lack a ProSys gene and Sys perception. In transgenic tobacco expressing SR-160 mRNA constitutively, the Sys receptor is membrane localized, binds Sys and activates early defense responses (Scheer et al. 2003). This is the first example of a protein receptor in plants having a dual function in defense and development. Ala substitution and deletion analyses of the Sys polypeptide indicated that the entire 18 amino acids are necessary for full biological activity, and that the N-terminal region might be important for recognition/binding to the Sys receptor, while the C-terminalresidues are more essential for activity (Pearce et al. 1993; Meindl et al. 1998). Upon binding to its receptor, Sys activates a complex cascade of signaling events leading to the activation of early (signaling-related) and late (defense-related) wound response genes, and the production of H2O2 (Ryan 2000). In tomato leaves, Sys causes a rapid (within minutes) depolarization of the plasma membrane in mesophyll cells, the alkalinization of the extracellular medium or cell wall apoplast, transient increases in cytoplasmic free Ca2+ concentrations, the activation of a mitogen-activated protein kinase (MAPK), and the release of linolenic acid (18:3 fatty acid) from cell membranes by the activation of a phospolipase A2 , leading to the biosynthesis of OPDA and JA through the octadecanoid pathway (Ryan 2000).

15.2.4 Systemin and the Systemic Wound Signal The plant wound defense response against insects or herbivore attacks, discovered by Green and Ryan (1972), is characterized by the accumulation of defense chemicals not only at the site of damage (local response), but also in other undamaged parts of the plant (systemic response). This implies that ‘wound’ signal(s) are generated at the sites of damage to activate the transcription of defense genes and the synthesis of antinutritional compounds throughout the plant (Green and Ryan 1972). Sys has the characteristics of a systemic wound signal. When radiolabeled Sys is placed in wounds made on leaves, Sys is loaded into the phloem and transported systemically throughout the plants within a few hours, in a similar fashion as the transport of 14 C-sucrose (Pearce et al. 1991; Narváez-Vásquez et al. 1994, 1995). In addition, the overexpression of the ProSys precursor gene under the regulation of the CaMV 35S promoter in transgenic tomato plants led to the constitutive accumulation of high levels of several defensive proteins in leaves (McGurl et al. 1994; Bergey et al. 1996). The ProSys overexpressing plants produced a graft-transmissible signal that activates defense genes in a wild type scion (McGurl et al. 1994). Conversely, when the 35S:ProSys transgene was expressed in the antisense orientation in transgenic tomato plants, the systemic activation of wound-inducible defense genes was strongly reduced and the plants were more susceptible to insect damage (McGurl et al. 1992; Orozco-Cárdenas et al. 1993). Thus,

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the cumulative results supported the hypothesis that Sys is an important component of a mobile systemic signal. Recent genetic evidence, however, indicated that JA or a JA-derived oxylipin, functions as the systemic wound signal in tomato, and that Sys main role is to up-regulate JA biosynthesis (Howe 2005). This was demonstrated using reciprocal grafting experiments with tomato mutants deficient in JA biosynthesis and JA perception, which were also defective in systemic wound signaling (Howe 2005; see also Howe and Schaller this volume). Accordingly, several enzymes of the octadecanoid pathway have been localized within the vascular bundle cells of tomato leaves, including LOX, AOS, and AOC (Wasternack et al. 2006). Tomato AOC is a rate-limiting enzyme required for wound-induced JA biosynthesis during Sys signaling (Stenzel et al. 2003). Therefore, Sys synthesis and JA synthesis have now been associated with the phloem. Altogether, the results of the grafting and localization studies indicated a new role for both Sys and JA in the amplification of the systemic signaling process as they are translocated through the plant vasculature (Ryan and Moura 2002; Howe 2005; Wasternack et al. 2006).

15.3 Hydroxyproline-Rich Systemin Glycopeptides (HypSys) The search for a functional homologue of Sys in tobacco, using a cell alkalinization assay (Felix and Boller 1995), resulted in the identification of two bioactive 18-aa glycopeptides in purified HPLC fractions obtained from tobacco leaf extracts (Pearce et al. 2001). The isolated peptides (called TobHypSys I and II) were capable of inducing the medium alkalinization of tobacco and tomato cell suspension cultures, and also of activating a 48-kDa MAP kinase and defense gene expression when supplied to excised tobacco leaves at nM concentrations (Pearce et al. 2001). TobHypSys I and II are hydroxyproline-rich glycopeptides, decorated with nine and six pentose units respectively (Table 15.1). The glycosyl moieties are required for full biological activity. TobHypSys I and II are derived from a single polyprotein precursor of 165 amino acids that has a signal peptide (Pearce et al. 2001), representing the first example in plants of multiple signaling peptides derived from the same protein precursor, a common feature found in animals (Tanaka 2003). Three HypSys glycopeptides were subsequently purified from tomato leaves, using the same medium alkalinization assay with L. peruvianum suspension cell cultures (Pearce and Ryan 2003). Tomato HypSys I, II, and III of 20, 18, and 15 residues respectively, are also derived from a single precursor protein of 146 amino acids, which is likely to be secreted by virtue of a predicted signal peptide (Fig. 15.1C). The tomato glycopeptides have between 8 and 16 pentose units, which appear to be arabinose residues bonded to hydroxyproline residues (Table 15.1; Pearce and Ryan 2003). The amino acid sequences of TomHypSys II and III peptides are very similar, with nine identical amino acids (Table 15.1), suggesting they have resulted from gene duplication and elongation events similar to ProSys.


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The presence of a signal peptide in both the tobacco and tomato HypSys precursors and the post-translational hydroxylation of proline and glycosylation indicates that the bioactive peptide precursors are synthesized through the secretory pathway. Immunolocalization studies using affinity-purified antibodies that only recognize epitopes in the tomato ProHypSys precursor indicates that the proprotein is localized in the cell walls of leaf vascular parenchyma cells (Fig. 15.2B; Narváez-Vásquez et al. 2005). Multiple HypSys glycopeptide signals derived from single protein precursors have also been found in other species of the Solanaceae, including petunia, nightshade, and potato (Pearce et al. 2006). In both tobacco and tomato, the ProHypSys genes are upregulated in the leaves by wounding, Sys, MeJA, and the HypSys peptides themselves (Pearce et al. 2001; Pearce and Ryan 2003). The Tobacco ProHypSys gene is also induced by ABA, chewing and phloem-feeding insects (Rocha-Granados et al. 2005). In tomato, low constitutive levels of HypSys mRNAs are found in leaves, but the basal levels are higher in other organs, including roots (Narváez-Vásquez and Ryan, unpublished results). Overexpression of the native HypSys genes in tobacco (Ren and Lu 2006) and tomato (Narváez-Vásquez et al. 2007) caused the constitutive upregulation of both signaling and defenserelated genes in the transgenic plants, similar to wounding and Sys in tomato. The 35S:HypSys transgenic tobacco plants were more resistant to insect attacks (Ren and Lu 2006). In addition, downregulation of TomHypSys gene expression by antisense technology also diminished the systemic wound inducibility of defense genes, suggesting that Sys and HypSys work cooperatively to upregulate the systemic wounddefense response in tomato (Narváez-Vásquez et al. 2007). The processing events responsible for the release of the HypSys peptides in the cell wall in response to wounding are still unknown. The presence of the –N/GR sequence at the N-termini of tobacco and tomato HypSys peptides suggests that these amino acids may be substrates for specific proteases in both species (Table 15.1). Wounding may cause intracellular proteases to mix with the precursor of HypSys peptides in the cell wall matrix as a consequence of cell damage. Wound- or Sys-induced ion fluxes and pH changes in leaf cells may as well induce secretion and/or activation of processing enzymes in the extracellular matrix (Schaller and Oecking 1999; Tanaka 2003). Alternatively, pathogen-derived peptidases may also be involved in processing of bioactive peptides leading to the activation of defense signaling cascades. In any event, the proteolytic release of multiple peptide signals from polyprotein precursors located in the cell walls may be among the plant earliest events in response to insect and pathogen attacks.

15.4 Arabidopsis Defense-Related Peptides (AtPeps) AtPep1 is the first endogenous defense-related peptide purified from leaves of Arabidopsis thaliana (Huffaker et al. 2006a). The AtPep1 peptide has 23 amino acids and is derived from the carboxy terminus of a signal-less 92-amino acid precursor protein (ProAtPep1; Fig. 15.1D). AtPep1 regulates the expression of defense genes

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induced by pathogens through the octadecanoid signaling pathway. The gene encoding ProAtPep1 is part of a small family of at least seven members in Arabidopsis, with orthologs in species of many other families, including important crop plants (Table 15.2). The size range of ProAtPep1-related precursor proteins reported to date is between 75 and 154 amino acids (Table 15.2). Based on their chromosomal location the Arabidopsis AtPep1 gene family has been classified in three subfamilies. Members of subfamilies I (ProAtPep1, 2, and 3) and II (ProAtPep4, 5, and 7) are grouped in two different locations in Chromosome V, and only one gene (ProAtPep6) belongs to the subfamily III located on chromosome II (Huffaker et al. 2006b). The Arabidopsis paralogs share the 10-amino acid motif SSG(K/R)xGxxN that appear to be important for full biological activity. Notably, Gly17 is the only conserved amino acid in all the other known AtPep1 orthologs (Table 15.2). Ala-substitution studies indicated that Gly17 is essential for AtPep1 biological activity (Huffaker et al. 2006b). In Arabidopsis, the ProAtPep1 gene is expressed at low constitutive levels in all tissues, with higher basal levels observed in flowers and roots (Huffaker et al. 2006a). In leaves, the ProAtPep1 gene is induced by wounding, MeJA and ethylene. Other gene family members show similar expression patterns in a wide range of tissues, and appear to be upregulated by MeJA (Huffaker et al. 2006a, b). Analysis of microarray gene expression data in Arabidopsis indicated that some of the ProAtPep1 paralogs are differentially regulated by bacterial and fungal pathogens and pathogen-derived elicitors (Huffaker et al. 2006a). AtPep1 supplied to excised Arabidopsis leaves through the cut petioles causes the activation of several genes involved in pathogen defense, such as those encoding for defensin (PDF-1.2), other pathogenesis-related (PR) proteins, ProAtPep1, and some of the ProAtPep1 paralogs (Huffaker et al. 2006b). Feeding AtPep1 to leaves also caused the production of H2 O2 in the vascular bundles (Huffaker et al. 2006a). When the ProAtPep1 gene was overexpressed in Arabidopsis under the regulation of the 35S promoter, the pathogen-related defensin gene was constitutively expressed and the transgenic plants were more resistant to infection by the soil borne pathogen Pythium irregulare (Huffaker et al. 2006a). Infected wild-type control plants grew smaller, especially their roots, than the transgenic plants. This result indicates that the overproduced ProAtPep1 has been processed to release the bioactive peptide to constitutively activate the pathogen-defense response in the transgenic tissue (Huffaker et al. 2006a). No endogenous peptides have been reported before that could activate genes associated with innate immunity in plants. An AtPep1-binding protein (∼170-kD) was isolated from microsomal membranes of Arabidopsis suspension cultured cells by photoaffinity labeling (Yamaguchi et al. 2006). The protein binds an iodinated-AtPep1 analog with a Kd of 0.25 nM, similar to the binding constant of the iodinated Sys analog to its SR-160 receptor (Kd of 0.17 nM; Scheer and Ryan 1999). Mass spectroscopy of tryptic digests identified the binding protein as a receptor-like protein kinase of 1123 amino acids, encoded by the gene At1g73080 in Arabidopsis (Yamaguchi et al. 2006). The AtPep1 receptor protein, called AtPepR1, contains 27 leucinerich repeats, a transmembrane region, and an intracellular Ser/Thr protein kinase

Precursor gene code

At5g64900 At5g64890 At5g64905 At5g09980 At5g09990 At2g22000 At5g09975 CD816645 CV505388 CV23975 BI311441 CD401281 CF333408 AK111113

Peptide

AtPep1 AtPep2 AtPep3 AtPep4 AtPep5 AtPep6 AtPep7 Canola Potato Poplar Medicago Soybean Rice1 Rice2

69-ATKVKAKQRGKEKVSSGRPGQHN∗ 74-DNKAKSKKRDKEKPSSGRPGQTNSVPNAAIQVYKED∗ 73-EIKARGKNKTKPTPSSGKGGKHN∗ 55-GLPGKKNVLKKSRESSGKPGGTNKKPF∗ 59-SLNVMRKGIRKQPVSSGKRGGVNDYDN∗ 53-ITAVLRRRPRPPPYSSGRPGQNN∗ 81-VSGNVAARKGKQQTSSGKGGGTN∗ 74-VARLTRRRPRPP-YSSGQPGQIN∗ 93-PTERRGRPPSRPKVGSGPPPQNN∗ 94-DAAVSALARRTPPVSRGGGGQTNTTTS∗ 87-LSSMGRGGPRRTPLTQGPPPQHN∗ 93-ASLMATRGSRGSKISDGSGPQHN∗ 131-ARLRPKPPGNPREGSGGNGGHHH∗ 65-DDSKPTRPGAPAEGSGGNGGAIHTAASS∗

C-terminal amino acid sequence

92 109 96 81 86 75 104 95 116 121 111 115 154 93

Precursor length (amino acids)

Table 15.2 Comparison of the C-terminal amino acid sequences of AtPep1 paralogs and orthologs containing putative AtPep sequences in plants. The sequence of AtPep1 (23 residues) is aligned with the C-termini deduced from six paralogs and seven orthologs. The amino acids of the conserved SSGR/KxGxxN motif present in all AtPep1 paralogs are bold. Adapted from Huffaker et al. (2006a, b)

15 Defense-Related Peptide Signals 323

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domain. Radiolabeled AtPep1 analog did not bind to microsomal membranes from two insertional mutants of At1g73080, and tobacco suspension cells transformed with the AtPepR1 gene were capable of binding to AtPep1 and showed the typical alkalinization response. These results demonstrate that PepR1 is a functional receptor (Yamaguchi et al. 2006). Four out of six AtPep1 homologs synthesized based on the deduced C-terminal amino acid sequence of ProPep1 paralogs in Arabidopsis were biologically active in the alkalinization assay with Arabidopsis cells. The bioactive synthetic peptides were also able to compete with the radiolabeled AtPep1 analog for binding to PepR1. This result suggested that PepR1 might actually be the receptor for at least five of the homologous AtPep1 peptides possibly generated during pathogen attacks (Yamaguchi et al. 2006).

15.5 Evolutionary Considerations The discovery of genetically different defense-related peptide signals in the Solanaceae, acting through the same octadecanoid-signaling pathway prompted Prof. C.A. Ryan and collaborators to ‘broaden the definition of systemins to include any signaling peptide from plants that activates plant defensive genes’ (Ryan and Pearce 2003). Thus, this definition should be extended to the Arabidopsis AtPep peptides, which are also upregulated by wounding and pathogens to activate genes involved in pathogen defense through the octadecanoid pathway. AtPep orthologs are also present in the Solanaceae (Table 15.2; Huffaker et al. 2006a). There is no sequence homology between the protein precursors of Sys, HypSys, and AtPep1 peptides (Fig. 15.1). The Sys and HypSys peptides have only been found in species of the Solanaceae, although the systemic wound defense response is ubiquitous in plants. Even the tomato and tobacco HypSys precursors only share a limited amino acid identity (compare Fig. 15.1B and C). However, the bioactive peptides show some degree of conservation suggestive of a common origin. For instance, they are enriched in proline/hydroxyproline, serine, and threonine residues, flanked by charged amino acids (Table 15.1). The HypSys glycopeptides contain – OOS- motifs, which are posttranslational modifications derived from –PPS- primary translation motifs also present in Sys (Table 15.1). AtPep1 lacks this motif, but some AtPep1 paralogs and orthologs are proline-rich and highly charged peptides (Table 15.2). Proline residues are known to confer structural conformations in the backbone chains of bioactive peptides that are important for the interactions of peptide ligands with their receptors (Rath et al. 2005). Like tomato Sys, AtPep1-related peptides are synthesized from larger precursor proteins that lack a classical signal sequence. In addition, the AtPep precursors are highly charged proteins, with stretches of lysine (K) and glutamic acid (E) residues that resemble the KEKE-like motifs (Realini et al. 1994), also found in ProSys (compare Fig. 15.1A and D). Because of this, and the similarities mentioned above, it has been proposed that peptide signals that activate defense responses against pathogen and herbivore attacks may have evolved from a common ancestor gene (Ryan and


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Pearce 2003; Huffaker et al. 2006a, b). Mutational events, such as gene insertions, followed by duplication and elongation events appear to be involved in the evolution of peptide signal precursors in plants (Ryan and Pearce 2003). This might explain why the prohormone precursors of defense related peptides have diverged more quickly than the actual bioactive peptides. Since AtPep1 peptides are widely spread in the plant kingdom, they may have evolved prior to the systemins in the Solanaceae. Since HypSys peptides are found in most species of the Solanaceae, and Sys only in the Solaneae subtribe, HypSys peptides appear to have evolved earlier than Sys. It is possible that the brassinolide receptor evolved initially and then was later recruited to facilitate systemin signaling. It would be interesting to see whether the HypSys peptides interact with homologs of the systemin receptor or have entirely different receptors. Notably, overexpression of the tomato ProSys gene in transgenic potato causes the accumulation of developmentally regulated proteins in the tubers (Narváez-Vásquez and Ryan 2002). The link between signaling pathways involved in plant development and defense is not well understood yet.

15.6 Perspectives The evidence suggests that defense-related peptide hormones play a primary role in the amplification of signals generated by insects and pathogen attacks in plants through the activation of a lipid-based signaling pathway in the phloem. The main purpose being the rapid systemic accumulation of deterrents and antinutritional factors throughout the plant that will diminish food quality, slow down growth, and/or prevent the colonization of invading organisms. This peptide-based signaling mechanism for plant defense is analogous to the activation of the inflammatory and acute-wound defense response mediated by peptide cytokines such as the interleukins and TNF-␣, through the eicosanoid signaling pathway in animals (Bergey et al. 1996). Although thoroughly studied in animal systems and yeast, the regulation of synthesis, processing, storage and secretion of peptide signals in plants is poorly understood. The processing sites that release the active peptides are not known, nor is the nature of the peptidases involved. The mechanism by which Sys and AtPep1 are released into the cell wall apoplast is still unknown. About half a dozen families of plant peptide hormones identified to date are synthesized from precursors lacking a signal sequence (Matsubayashi and Sakagami 2006), suggesting that more often than in animals and yeast, plant regulatory peptides are processed and transported through non-classical mechanisms to the extracellular space (Nickel 2003). Further studies on the biogenesis of defense-related peptide hormones, and the discovery of new peptide signals and their receptors in plants, will probably provide more evidence for the evolution of defense peptide signaling systems in eukaryotes from a common ancestor.

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Narváez-Vásquez J, Orozco-Cárdenas ML, Ryan CA (1994) A sulfhydryl reagent modulates systemic signaling for wound-induced and systemin-induced proteinase-inhibitor synthesis. Plant Physiol 105:725–730 Narváez-Vásquez J, Orozco-Cárdenas ML, Ryan CA (2007) Systemic wound signaling in tomato leaves is cooperatively regulated by multiple plant peptides. Plant Mol Biol 65:711–718 Narváez-Vásquez J, Pearce G, Orozco-Cárdenas ML, Franceschi VR, Ryan CA (1995) Autoradiographic and biochemical-evidence for the systemic translocation of systemin in tomato plants. Planta 195:593–600 Narváez-Vásquez J, Pearce G, Ryan CA (2005) The plant cell wall matrix harbors a precursor of defense signaling peptides. Proc Natl Acad Sci USA 102:12974–12977 Narváez-Vásquez J, Ryan CA (2002) The systemin precursor gene regulates both defensive and developmental genes in Solanum tuberosum. Proc Natl Acad Sci USA 99:15818–15821 Narváez-Vásquez J, Ryan CA (2004) The cellular localization of prosystemin: a functional role for phloem parenchyma in systemic wound signaling. Planta 218:360–369 Nickel W (2003) The mystery of nonclassical protein secretion – A current view on cargo proteins and potential export routes. Eur J Biochem 270:2109–2119 Orozco-Cárdenas ML, McGurl B, Ryan CA (1993) Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae. Proc Natl Acad Sci USA 90:8273–8276 Pautot V, Holzer FM, Reish B, Walling LL (1993) Leucine aminopeptidase: an inducible component of the defense response in Lycopersicon esculentum (tomato). Proc Natl Acad Sci USA 90:9906–9910 Pearce G, Johnson S, Ryan CA (1993) Structure-activity of deleted and substituted systemin, an 18-amino acid polypeptide inducer of plant defensive genes. J Biol Chem 268:212–216 Pearce G, Moura DS, Stratmann J, Ryan CA (2001) Production of multiple plant hormones from a single polyprotein precursor. Nature 411:817–820 Pearce G, Narváez-Vásquez J, Ryan CA (2006) Systemins. In: Kastin AJ (ed) Handbook of biologically active peptides. Academic Press, San Diego, pp 49–53 Pearce G, Ryan CA (2003) Systemic signaling in tomato plants for defense against herbivores – isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene. J Biol Chem 278:30044–30050 Pearce G, Strydom D, Johnson S, Ryan CA (1991) A polypeptide from tomato leaves activates the expression of proteinase inhibitor genes. Science 253:895–898 Rath A, Davison AR, Deber CM (2005) The structure of ‘unstructured’ regions in peptides and proteins: role of the polyproline II helix in protein folding and recognition. Biopolymers 80: 179–185 Realini C, Rogers SW, Rechsteiner M (1994) KEKE motifs – proposed roles in protein–protein association and presentation of peptides by MHC Class-I receptors. FEBS Lett 348:109–113 Ren F, Lu Y-T (2006) Overexpression of tobacco hydroxyproline-rich glycopeptide systemin precursor A in transgenic tobacco enhances resistance against Helicoverpa armigera larvae. Plant Sci 171:286–292 Rocha-Granados MC, Sánchez-Hernández C, Sánchez-Hernández C, Mart´ınez-Gallardo NA, Ochoa-Alejo N, Délano-Frier JP (2005) The expression of the hydroxyproline-rich glycopeptide systemin precursor A in response to (a)biotic stress and elicitors is indicative of its role in the regulation of the wound response in tobacco (Nicotiana tabacum L.). Planta 222:794–810 Rojo E, Martin R, Carter C, Zouhar J, Pan SQ, Plotnikova J, Jin HL, Paneque M, Sanchez-Serrano JJ, Baker B, Ausubel FM, Raikhel NV (2004) VPE gamma exhibits a caspase-like activity that contributes to defense against pathogens. Curr Biol 14:1897–1906 Ryan CA (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochim Biophys Acta 1477:112–121 Ryan CA, Moura DS (2002) Systemic wound signalling in plants: a new perception. Proc Natl Acad Sci USA 99:6519–6520

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Chapter 16

MAP Kinases in Plant Responses to Herbivory Johannes Stratmann

Mitogen-activated protein kinases (MAPKs) are signal transducing enzymes involved in many plant responses to the environment including defense responses to herbivory. Phytophagous insects generate various plant- and insect-derived primary signals such as the plant signaling peptide systemin, mechanical wound signals, and fatty acid-amino acid conjugates. These signals activate a set of specific MAPKs. Reducing the function of these MAPKs impairs the synthesis of secondary stress signals including the key wound signal jasmonic acid, which is required for the upregulation of defense proteins. Consequently, loss-of-MAPKfunction results in reduced resistance of plants to herbivorous insects. Insectresponsive MAPKs also function as essential signaling relays in defense responses to pathogens and abiotic stress. It is a major challenge for future research to unravel the molecular mechanisms that perceive and integrate stress signals to generate stimulus-specific MAPK signaling patterns which result in specific cellular defense responses.

16.1 Introduction A wealth of genomic information in all eukaryotes has revealed the preeminent role of protein kinases for the regulation of cellular processes. PlantsP, an interactive website dedicated to the study of plant phosphorylation, estimated that the Arabidopsis genome contains about 1000 putative protein kinases that constitute 5 major classes with a total of 80 distinct families (http://plantsp.genomics.purdue.edu /html). Unlike in animals and yeast, tyrosine-specific phosphorylation is rare in plants. Almost all plant protein kinases are Ser/Thr-specific kinases. They are structurally defined by the presence of 12 conserved subdomains that constitute the catalytic core of the enzyme. The kinase domain is often extended N- and C-terminally by other protein domains (Hanks and Hunter 1995). Many protein kinases become J. Stratmann Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA e-mail: [email protected]


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activated by phosphorylation via other kinases and inactivated by dephosphorylation via protein phosphatases, which comprise about 300 members in the Arabidopsis genome (PlantP). Reversible phosphorylation is known to be a part of many signaling pathways that regulate developmental processes and plant responses to the environment. This chapter reviews the role of mitogen-activated protein kinase (MAPK) cascades in plant responses to herbivorous insects and mechanical wounding. To understand the function of MAPKs, it is important to note that many plant stress responses overlap at various levels of signal transduction and cellular responses. Global transcriptome analyses revealed overlaps among gene expression profiles induced by different stress stimuli, but also stimulus-specific gene expression (Cheong et al. 2002; Reymond et al. 2000; Schenk et al. 2000). It appears that a limited number of secondary stress signals, such as ion fluxes (e.g., Ca2+ and H+ ), jasmonic acid (JA), ethylene, reactive oxygen species (ROS), and salicylic acid (SA), function in multiple signaling pathways that can be induced by structurally and functionally diverse stress signals. MAPKs are known to function as convergence points for multiple stress signals. The interaction and coordination of these signaling components is thought to occur in a signaling network where multiple signaling elements are highly interconnected and mutually affect each other. While this review will focus on signal transduction in plant responses to herbivory, it will take into account that wound-responsive MAPKs also function in other stress signaling pathways. Only very few studies address the role of MAPKs and other protein kinases in response to damage inflicted by herbivorous insects. Most studies have focused on the role of protein kinases in the response to leaf wounding with mechanical devices, such as a hemostat or forceps for crushing, razor blades for cutting, or carborundum for leaf surface abrasion. It should be noted that mechanical wounding and wounding by chewing insects result in the generation of several primary wound signals, such as systemically propagated rapid electrical and hydraulic signals (Malone 1996; Rhodes et al. 1996; Stratmann and Ryan 1997; Wildon et al. 1992), and peptides such as systemin and hydroxyproline-rich systemins (Pearce et al. 1991, 2001; Pearce and Ryan 2003). In addition, insects deliver fatty acid-amino acid conjugates (Alborn et al. 1997; Halitschke et al. 2003) or other insect-specific elicitors like ␤-glucosidase (Mattiacci et al. 1995) to wound sites which function as elicitors of direct and indirect defense responses. For some insect species, however, the mechanical stimuli produced by insect mandibles alone may be sufficient to trigger a full-fledged wound response and resistance to insects (Mithofer et al. 2005). Furthermore, unlike wounding by insects, wounding with mechanical devices causes desiccation stress in the wounded leaf with concomitant activation of water stress-related responses (Birkenmeier and Ryan 1998; Reymond et al. 2000). If protein kinases are activated as a consequence of wounding with mechanical devices, it is unclear what the actual activating signal is and whether this is relevant to wound responses elicited by insects. Chewing insects represent only one feeding guild among phytophagous insects. With regard to the mechanics of plant damage, they do not have much in common with sucking-piercing insects such as aphids, whiteflies, and thrips. This group

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of insects employs sophisticated strategies to imbibe cell or phloem contents. The actual wound stimulus is more confined and is produced by elaborate specialized stylets that can penetrate single cells. However, sucking-piercing insects seem to activate defense pathways that are also induced by microbial pathogens, nematodes, and chewing insects (Li et al. 2002b, 2006; reviewed in Walling 2000). Protein phosphatases remove phosphate groups from MAPKs and thereby inactivate them. With a few exceptions (Meskiene et al. 1998; Seo et al. 2007; Xu et al. 1998), not much is known on the role of protein phosphatases in responses to wounding and herbivory. This is also true for the protein substrates of MAPKs and the upstream kinases that activate MAPKs. Therefore, these groups of proteins will not be discussed in detail in this chapter, even though they are of utmost importance for understanding the role and function of MAPKs.

16.2 MAP Kinases in Plant Responses to Herbivory 16.2.1 Mitogen-Activated Protein Kinase (MAPK) Cascades A MAPK cascade is a three-tiered phosphorelay cascade which is initiated by a MAPK kinase kinase (MAPKKK, also known as MEKK). MAPKKKs are often activated as a consequence of receptor-mediated perception of stress signals. They phosphorylate and thereby activate MAPK kinases (MAPKKs, also known as MEKs or MKKs) on two Ser/Thr residues in the kinase activation loop. MAPKKs are dualspecificity kinases which activate MAPKs (also known as MPKs) through phosphorylation of a Thr and a Tyr residue in a TE/DY phosphorylation motif located in the MAPK activation loop between kinase subdomains VII and VIII (Ichimura et al. 2002). In plants, MAPKKs are the only well-known protein kinases that can phosphorylate Tyr residues. Some MAPKs are transcriptionally regulated, but posttranslational activation of preformed MAPKs via phosphorylation often precedes transcriptional activation and is required for kinase activity. This might explain why a microarray analysis found only a relatively small number of kinase transcripts upregulated in response to herbivory by a generalist and a specialist caterpillar in Arabidopsis plants (Reymond et al. 2004). MAPKs are Ser/Thr kinases which phosphorylate a range of cytosolic and nuclear substrate proteins that function in metabolism or transcription, at least in metazoan and yeast cells. In plants, not many in vivo substrates have been identified, but a picture is emerging that is consistent with animal models. Plant genomes contain a large number of MAPKs, e.g., 20 in Arabidopsis, 21 in poplar, and 15 in rice (Hamel et al. 2006), but only half the number of MAPKKs. The number of functional MAPKKKs is controversial (Champion et al. 2004) and may exceed 60 kinases from various gene families. The general function and genome representation of MAPK cascade components has been described in excellent reviews (Champion et al. 2004; Hamel et al. 2006; Ichimura et al. 2002) and will not be covered here.

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Phylogenetic comparisons of plant MAPKs revealed two major clades, one characterized by a TEY, and the other one by a TDY phosphorylation motif (Champion et al. 2004; Hamel et al. 2006; Ichimura et al. 2002). Among dicots, only members of the TEY clade are known to be wound-responsive. They fall into the three distinct subgroups A1, A2, and B1. None of these MAPKs is exclusively activated by wounding or wound signals. It is a general feature of all characterized MAPKs that they respond to a wide range of stimuli and signals (reviewed in Nakagami et al. 2005). Wound-responsive TDY MAPKs have also been identified in monocots. Possible differences between monocot and dicot MAPK signaling in the wound response were recently reviewed and will not be discussed here (Rakwal and Agrawal 2003).

16.2.2 SIPK and WIPK The best studied plant MAPKs are the stress-induced MAPK (SIPK) and the wound-induced MAPK (WIPK) from tobacco, and their orthologs in Arabidopsis, alfalfa, parsley, and tomato. They belong to the A2 and A1 subgroups, respectively. Most stress signals that activate SIPK also activate WIPK (reviewed in Nakagami et al. 2005; Zhang and Klessig 2000). SIPK activity increases in response to almost any disturbance in the cellular environment, including biotic and abiotic stimuli, and even seemingly innocuous stimuli like touch (Nakagami et al. 2005). SIPK and orthologs are preformed and activated posttranslationally without concomitant changes in transcript levels (Higgins et al. 2007; Ichimura et al. 2000; Zhang and Klessig 1998). In contrast, WIPK and orthologs are transcriptionally and posttranslationally regulated in response to wounding and other stress signals. However, increases in transcript levels do not always result in higher WIPK protein levels. Whether this is due to increased protein turnover is presently unclear (Bögre et al. 1997; Higgins et al. 2007; Hirt 1999; Seo et al. 1999; Zhang and Klessig 2000). Detailed analyses of MAPKs activated by wounding or wound signals are only available for WIPK and SIPK in tobacco and their orthologs in tomato. Leaf wounding, stem excision, and feeding by Manduca sexta (Lepidoptera) larvae as well as the wound signals systemin, hydroxyproline-rich systemins, oligosaccharides, and a novel wound-inducible diterpene (11E,13E)-labda-11,13-diene-8␣,15-diol, all induced MAPK activity in tobacco and tomato (Higgins et al. 2007; Holley et al. 2003; Seo et al. 1995, 2003, 2007; Stratmann and Ryan 1997). In response to wounding and insect attack, the MAPK activity increased systemically, at least over short distances (Seo et al. 1999; Stratmann and Ryan 1997). The function of systemic increases in MAPK activity is not known. Seo et al. (1995) presented the first loss-of-function analysis of a plant MAPK. In wild type tobacco plants, wounding leads to increases in JA levels followed by expression of basic pathogenesis-related (PR) genes and proteinase inhibitor II (PI-II). Surprisingly, in a transgenic line in which the WIPK gene was cosuppressed, wounding induced salicylic acid (SA) synthesis and expression of acidic PR genes,


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while JA synthesis and expression of basic PR genes and PI-II was suppressed. This is characteristic of defense responses to microbial pathogens. Since responses to pathogens and wounding are often mutually exclusive (Felton et al. 1999), the cosuppression results suggested that WIPK is participating in a switch-like mechanism that determines whether the wound response or the response to pathogens will be expressed. To further test the role of WIPK for JA synthesis in response to wounding, transgenic tobacco plants were generated that overexpressed WIPK (Seo et al. 1999). Surprisingly, unwounded plants exhibited constitutively elevated WIPK activity (1.5 fold), 3–4 fold higher JA levels as compared to control plants, and constitutive expression of the PI-II gene. These results raise several questions. How does overexpression lead to increased kinase activity in the absence of wounding? Were the upstream kinases (MAPKKs and MAPKKKs) also activated? The authors suggested that overexpression may lead to autoactivation of WIPK, which is a well-known phenomenon for recombinant MAPKs. The results were questioned by Kim et al. who showed that expression of a constitutively active mutant of NtMEK2, the common MAPKK for SIPK and WIPK (Yang et al. 2001), leads to increased activity of both MAPKs and ethylene synthesis (Kim et al. 2003). However, they were not able to detect increases in JA levels. These differences may be explained by the different gain-of-function approaches; overexpression of a constitutively active MAPKK which activates at least two MAPKs, versus overexpression of WIPK. In any case, the conflicting data indicate that it is important to complement gain-of-function with loss-of-function studies. In their most recent study, the Ohashi group employed the RNA interference (RNAi) technique to silence WIPK and SIPK both individually and simultaneously (Seo et al. 2007). SIPK has an almost identical paralog, Ntf4, which has recently been shown to be largely functionally redundant with SIPK, at least with regard to pathogenesis-related responses (Ren et al. 2006). The RNAi construct that targeted SIPK also silenced Ntf4. These transgenic plants exhibited normal growth and development, but reduced wound-induced JA levels. Wound-induced ethylene synthesis and accumulation of transcripts for the JA-biosynthetic enzymes ALLENE OXIDE SYNTHASE (AOS) and ALLENE OXIDE CYCLASE (AOC) were at least partially SIPK-dependent. Wounding of SIPK-silenced plants also resulted in higher levels of acidic PR transcripts, a phenomenon also observed in the WIPK-cosuppressed line (Seo et al. 1995). But WIPK-RNAi lines did not produce this response. Furthermore, SA levels, which were increased in the WIPK-cosuppressed lines, were not increased in either wounded WIPK-RNAi or SIPK-RNAi lines, but they went up in SIPK/WIPK-cosilenced plants. A significant reduction in wound response marker genes was not observed in any of the three RNAi lines. Taken together, the three papers from Ohashi’s group point to the following scenario. In response to wounding, JA and ethylene levels increase in a SIPK- and/or WIPK-dependent manner. Since silencing of the kinases was strong, but the reduction of JA and ethylene levels only partial, additional regulatory mechanisms may be in place. To reconcile conflicting data, the authors suggested that either SIPK or WIPK regulate the synthesis of an unknown wound-induced inhibitor which may

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act together with JA to suppress SA synthesis. Is the inhibition of JA synthesis in the silenced tobacco plants a consequence of cross-inhibition by SA, a known inhibitor of JA synthesis and signaling (Doares et al. 1995)? The authors point out that this is unlikely since silencing of SIPK and WIPK alone did not result in SA synthesis, yet the JA levels were much lower than in control plants. It remains a challenge to reconcile the different results obtained by using RNAi or cosuppression of WIPK. Since WIPK and SIPK are convergence points of many stress signals that function in a signaling network, the ratio of SIPK to WIPK levels, which probably differs among the various MAPK-silenced lines, may be important to generate appropriate responses (see 16.3 Perspectives).

16.2.3 LeMPK1, LeMPK2, and LeMPK3 In tomato, the wound response is mediated by the wound signaling peptide systemin (Narváez-Vásquez and Orozco-Cárdenas this volume). Systemin is both necessary and sufficient for the successful defense of tomato plants against herbivorous insects as shown by antisense inhibition and prosystemin overexpression experiments (Orozco-Cardenas et al. 1993; Chen et al. 2005; McGurl et al. 1994). In response to leaf wounding or the application of systemin through cut stems, MAPK activity increased within minutes in a systemic manner. The stem excision itself rapidly induced MAPK activity in the leaves indicating that a rapidly propagated mechanical signal, probably of hydraulic nature, caused the systemic MAPK activation (Stratmann and Ryan 1997). Stem excision alone induced a transient increase in MAPK activity which lasted only ∼15 min. In the presence of systemin, MAPK activity was stronger and more prolonged (>60 min). Activation of MAPKs by systemin was receptor-dependent. A systemin analog that binds to the systemin receptor SR160 without activating it (Meindl et al. 1998; Scheer and Ryan 1999) prevented systemin-induced MAPK activation (Stratmann and Ryan 1997). In addition, the naphthylurea compound suramin, a known inhibitor of cytokine–cytokine receptor interactions in animals, was shown to prevent binding of systemin to SR160 and blocked MAPK activation (Stratmann et al. 2000a). Systemin- and wound-induced MAPK activation was not altered in the JA-deficient mutant def1, indicating that MAPKs function either upstream of JA synthesis or in a parallel pathway. Systemininduced MAPK activity and PI-II synthesis exhibited the same dose-response curve, indicating that MAPKs are functioning in the signaling pathway that leads to expression of wound response genes (Stratmann and Ryan 1997). Later, it was shown that systemin and wounding activate three MAPKs, called LeMPK1, LeMPK2, and LeMPK3 (for Lycopersicon esculentum MAPK), which represent the tomato orthologs of the tobacco SIPK, Ntf4, and WIPK, respectively (Higgins et al. 2007; Holley et al. 2003). Wounding generates various signals, and application of systemin through cut stems involves a wound stimulus. To study systemin-specific responses, we used transgenic plants that overexpress prosystemin under the control of the 35S promoter.


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These plants exhibit constitutive accumulation of proteinase inhibitors (McGurl et al. 1994) and had been used earlier to identify JA biosynthetic and signaling mutants as suppressors of prosystemin-mediated responses (spr mutants; Howe and Ryan 1999). Silencing the LeMPK1, LeMPK2, and LeMPK3 genes in these transgenic plants by virus-induced gene silencing (VIGS) resulted in reduced levels of defensive proteinase inhibitors. This demonstrated that the function of these MAPKs is required for prosystemin-induced gene expression. Cosilencing of LeMPK1/2 in 35S::Prosystemin plants also reduced JA synthesis in response to wounding, and complementation of LeMPK1/2-silenced plants with methyl-JA (MeJA) restored proteinase inhibitor synthesis. These data support a role of LeMPK1/2 in regulating JA synthesis (Kaitheri Kandoth et al. 2007). They are consistent with experiments showing that MAPK activity does not increase after application of JA or its precursor 12-oxo-phytodienoic acid (Stratmann and Ryan 1997). Since it is well-established that the LeMPK3 ortholog WIPK is required for JA synthesis in tobacco (Seo et al. 1995, 1999, 2007), it is likely that systemin activates at least three MAPKs which function upstream of JA synthesis. LeMPK1 and 2 are 95% identical at the amino acid level, and they are activated in a concerted manner by a range of stress signals such as oligosaccharide elicitors, wounding, ultraviolet-B radiation, and the fungal toxin fusicoccin suggesting that they are functionally redundant (Higgins et al. 2007; Holley et al. 2003). Surprisingly, we found that they were both required for defense gene activation by systemin. VIGS of either LeMPK1 or LeMPK2 reduced proteinase inhibitor levels in 35S::Prosystemin plants (Kaitheri Kandoth et al. 2007). There is evidence that SIPK activates WIPK, possibly by regulating WIPK expression (Liu et al. 2003), but this is not consistent with our results in tomato. Since LeMPK1 and 2 appear to share the same functions in leaf tissue, gene dosage effects may be important for modulating signaling flow and thus cellular responses. Many MAPKs in various plant species evolved as a consequence of recent gene duplication events (Hamel et al. 2006). Most of them are expressed, but it is not understood why the presence of paralogs is adaptive. Additional gene knock-out experiments targeting paralogous MAPKs should help to elucidate this phenomenon. JA synthesis is initiated in the chloroplasts resulting in the synthesis of the JA precursor 12-oxo-phytodienoic acid which is then further metabolized in peroxisomes to JA (Schaller and Stintzi this volume). It is not known how the systemin signal is transduced from the plasma membrane-bound receptor kinase SR160 via the cytosol to the chloroplast. Most plant MAPKs are cytosolic proteins. Therefore, the LeMPKs are likely to further relay the systemin signal to activate JA biosynthetic processes in the chloroplasts. The identification of LeMPK substrates is essential to understand the highly compartmentalized wound/systemin response in tomato cells (Schilmiller and Howe 2005; Wasternack et al. 2006). Known substrates of the LeMPK1/2 ortholog AtMPK6 are the cytosolic ethylene-forming enzymes 1-aminocyclopropane-1-carboxylate synthase 2 and 6 (ACS2 and ACS6; Liu and Zhang 2004). AtACS2/6 were phosphorylated by AtMPK6 resulting in increased ACS activity and ethylene synthesis in response to the bacterial flagellin elicitor. Ethylene is also an essential second messenger in the wound response (O’Donnell et al. 1996)

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and is generated in response to systemin in suspension-cultured L. peruvianum cells (Felix and Boller 1995). In tobacco, wound-induced ethylene synthesis was partially regulated by SIPK (LeMPK1/2 ortholog). It is conceivable that JA synthesis is indirectly regulated by SIPK via ethylene. Based on remarkable parallels between the animal inflammatory response and the tomato wound response, it was proposed that a MAPK activates a cytosolic phospholipase A2 (cPLA2 ) which would initiate JA synthesis via production of linolenic acid from membrane lipids (Stratmann et al. 2000a). In animal cells, certain growth factors activate a MAPK which in turn activates cPLA2 via phosphorylation (Lin et al. 1993). A cPLA2 activity was identified in tomato leaf extracts in response to wounding and systemin (Narváez-Vásquez et al. 1999). However, a specific wound-responsive cPLA2 has not been identified, and it is now evident that the first steps of JA synthesis occur in the chloroplasts (Schilmiller and Howe 2005; Wasternack et al. 2006). This is difficult to reconcile with the activation of a cytosolic PLA2 by MAPKs. It is also not known from other model systems that MAPKs can translocate from the cytosol into organelles. Another intermediate signal in the wound response are reactive oxygen species (ROS), which are thought to function downstream of JA and are generated by a NADPH oxidase activity (Orozco-Cárdenas et al. 2001; Sagi et al. 2004). Since LeMPK1-3 function upstream of JA, they might also regulate ROS synthesis. ROS are known as activators of MAPKs (Kovtun et al. 2000) and might cause oxidative damage to signaling components that regulate MAPKs, e.g., protein phosphatases. But the exact mechanism of MAPK activation by ROS is not known. In response to fungal elicitors which trigger an oxidative burst, MAPKs are activated independently of ROS (Cazale et al. 1999; Ligterink et al. 1997; Romeis et al. 1999). Vice versa, there is evidence for a MAPK-independent oxidative burst in host-specific fungal-plant interactions (Romeis et al. 1999). Together this suggests that, at least in some signaling pathways, MAPK activity and ROS generation are induced in parallel and may function independently of each other. However, the relation of wound-induced ROS and MAPK activity remains to be resolved. Systemin perception by SR160 induces extracellular alkalinization (EA) and a concomitant membrane depolarization (Felix and Boller 1995; Moyen and Johannes 1996; Schaller and Oecking 1999; Stratmann et al. 2000a). The alkalinization response is not unique to systemin, but induced by almost any stress signal. However, it is not known what the consequences of the EA are for signal transduction. Reversing the proton gradient by activators of the plasma membrane proton ATPase (PMA) compromised systemin-induced gene expression suggesting that the PMA regulates the extracellular pH in response to systemin, and that EA is an essential signaling event for the systemin-induced wound response (Schaller and Oecking 1999). It has been proposed that systemin inactivates the PMA in a calcium-dependent manner via phosphorylation through a calcium-dependent protein kinase (Rutschmann et al. 2002; Schaller and Oecking 1999; Schaller 1999). The fungal toxin fusicoccin (FC), which permanently activates the PMA, caused extracellular acidification and inhibited the systemin-induced wound response. Moreover, FC application resulted in expression of PR-genes. This prompted Schaller and Oecking (1999) to propose


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that PMA activity functions as a switch that determines whether wound response genes or PR-genes will be expressed. Since most stress stimuli and signals that induce EA also activate A1- and A2MAPKs, we wondered whether EA and the consequences thereof are required for MAPK activation or vice versa. We performed a comparative analysis of pH effects and MAPK activity in response to systemin and fusicoccin. Fusicoccin activated the wounding/systemin-responsive LeMPK1, 2, and 3, showing that both extracellular alkalinization and acidification lead to the activation of the same MAPKs. However, the MAPK activation kinetics were distinct, with systemin causing transient and FC causing prolonged MAPK activity. Simultaneous application of FC and systemin did not lead to immediate pH changes but resulted in rapid increases in MAPK activity. To test whether changes in the extracellular pH can be uncoupled from MAPK activation, conditioned growth medium of L. peruvianum suspension-cultured cells was exchanged with fresh medium. This resulted in a strong medium acidification without concomitant MAPK activation. Together, these experiments demonstrated that changes in the extracellular pH are neither required nor sufficient for MAPK activation. It could also be excluded that MAPKs regulate EA. Therefore, systemininduced MAPK activation and EA most likely operate in parallel and are not part of one linear signaling pathway that induces wound response gene expression (Higgins et al. 2007). Systemin is an essential component in the tomato defense response against herbivorous insects and is required for the regulation of JA synthesis (see above). JA functions not only as an intracellular second messenger but also as the long-distance wound signal (Schilmiller and Howe 2005; Stratmann 2003). JA biosynthetic and signaling mutants are strongly impaired in the resistance to herbivorous insects (Howe et al. 1996; Li et al. 2002a, 2004). Consistent with a role of MAPKs in the regulation of JA synthesis, we showed that cosilencing of LeMPK1 and LeMPK2 severely compromised resistance to M. sexta larvae in 35S::Prosystemin plants. We also showed that Manduca sexta larvae systemically induce LeMPK1, LeMPK2, and LeMPK3 activity (Kaitheri Kandoth et al. 2007). The wound/systemin-responsive LeMPKs also play a role in resistance of tomato plants to sucking-piercing insects such as aphids. The mechanics of the damage inflicted by aphid stylets is different from chewing insects with regard to scale and nature. Resistance of tomato plants to aphids and certain whitefly and nematode species is dependent on the Mi-1 gene (Li et al. 2006). Mi-1-mediated resistance requires salicylic acid (SA) and results in upregulation of SA-inducible PR genes. This is reminiscent of tomato defense responses against pathogens which are regulated by MAPK cascades (Del Pozo et al. 2004; Ekengren et al. 2003; Pedley and Martin 2004). An involvement of MAPK(K)s in aphid resistance was tested by VIGS of LeMPK1/2, LeMPK3, and LeMKK2. All three VIGS constructs resulted in increased aphid survival on Mi-1-expressing tomato leaves demonstrating that Mi-1-mediated resistance to aphids is regulated by a MAPK cascade (Li et al. 2006). These two reports represent the only loss-of-function analyses showing that specific kinases function as essential signaling components in plant defense responses that confer resistance to herbivorous insects.

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As pointed out earlier, MAPKs of the A1 and A2 subgroup are highly promiscuous kinases responding to almost any disturbance in the cellular environment. LeMPK1 and 2 function as convergence points for stress stimuli and signals such as feeding insects, wounding, systemin, hydroxyproline-rich systemins, oligosaccharide elicitors, and ultraviolet-B and -C radiation (Holley et al. 2003; Higgins et al. 2007; Pearce et al. 2001; Stratmann et al. 2000b). The response of tomato leaves to ultraviolet-B (UV-B) radiation is interesting with regard to the specificity and interconnectedness of MAPK-regulated stress responses. While UVB alone strongly induced MAPK activation, it induced neither 12-oxo-phytodienoic acid and JA formation, nor proteinase inhibitor synthesis. However, a weak wound stimulus given prior to UV-B irradiation resulted in a synergistic systemic accumulation of proteinase inhibitors (Stratmann et al. 2000b). Since proteinase inhibitor gene expression is strictly JA-dependent, this experiment indicates that MAPK activation by UV-B is not sufficient to bring about JA synthesis and defense gene expression. Similar experiments with Nicotiana attenuata plants in a field setting demonstrated that UV-B-responsive genes show a strong overlap with insectinduced genes (Izaguirre et al. 2003). Taken together, it seems evident that activation of the same MAPKs by different stress signals and stimuli is associated with both signal-specific responses and overlaps among stress responses. No mechanism has been identified so far that would explain this phenomenon (see 16.3 Perspectives).

16.2.4 The Role of MPK4 in the Wound Response MAPKs of the A1 and A2 subgroups are not the only wound-responsive MAPKs. In Arabidopsis, the B1-MAPK, AtMPK4, can be activated by wounding, touch (gentle moving of the plants with hands) and some abiotic stresses (Ichimura et al. 2000). MAPK mutants are notoriously difficult to identify in standard mutant screens. Therefore, it was surprising that an mpk4 mutant could be identified (Petersen et al. 2000), especially considering that there is a closely related homolog, AtMPK11, in the same subclade, and MPK5 and MPK12 in two additional subclades of the B1 subgroup (Ichimura et al. 2002). The isolation of mpk4 also demonstrates that neither the other B1-MAPKs nor the multistress-responsive A-group MAPKs are able to compensate for the loss of MPK4. The mpk4 mutant is more resistant to bacterial and oomycete pathogens as compared to wild type plants. This is accompanied by constitutive expression of PR genes and elevated SA levels, which is indicative of the pathogen-induced systemic acquired resistance (SAR). SAR is a broad-spectrum resistance to a wide range of microbial pathogens. Mutant mpk4 plants were MeJA-insensitive, even in transgenic NahG-expressing plants that do not accumulate SA. This demonstrated that the JA insensitivity was not caused by SA – JA crosstalk. The mpk4 mutant showed a dwarf phenotype, but responded normally to multiple abiotic stress treatments and plant hormones. The effect of the mutation was specific as determined by a microarray analysis that found only a small number of genes whose expression was affected by the mutation.


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The insensitivity of mpk4 plants to JA suggests that MPK4 functions in JA signaling downstream of JA synthesis, unlike the A group MAPKs that most likely function upstream of JA synthesis. It will be interesting to unravel the relationship of MPK4 and the E3-ligase SCFCOI1 , which also functions downstream of JA and has recently been shown to target repressors of JA-induced gene expression for degradation by the proteasome (Chini et al. 2007; Thines et al. 2007). While Petersen et al. (2000) did not test whether the Arabidopsis mpk4 mutant shows a compromised wound response, this was studied in tobacco by Gomi et al. (2005). The putative tobacco ortholog NtMPK4 can be transiently activated by leaf wounding, similarly to A group MAPKs. NtMPK4-RNAi plants showed a phenotype that closely resembled the mpk4 phenotype in Arabidopsis with dwarfed morphology, elevated SA levels, and expression of SA-responsive PR genes. Wound-induced expression of the wound response marker gene PI-II was reduced, while AOS expression was not. JA levels were not measured in the NtMPK4-RNAi plants, and it was not tested whether PI-II expression can be restored in JA-treated NtMPK4-RNAi plants. It will be important to continue these studies to establish whether MPK4 is required for defenses against herbivorous insects, and how MPK4 acts together with the other wound-responsive MAPKs to orchestrate the wound response.

16.3 Perspectives We know surprisingly little about reversible phosphorylation in responses to feeding insects. The studies reviewed here point to a role of MAPKs in herbivory. But this conclusion is mostly based on experiments that apply mechanical wounding or wound signals such as systemin. MAPKs involved in responses to pathogens have gained much greater attention including multiple loss- and gain-of-function studies and detailed biochemical analyses. To my knowledge, there are only two MAPK loss-of-function studies that directly involve insects (Kaitheri Kandoth et al. 2007; Li et al. 2006). In the future, it will be important to study the role of each MAPK in greater detail by exposing genetically manipulated plants to herbivorous insects. Since different insect feeding guilds cause different types of damage to plants (see 16.1 Introduction), it will also be important to further delineate whether certain MAPKs function in defenses against all or only a limited subset of insect species. The loss-of-function studies regarding the tomato MAPKs showed that LeMPK1, 2, and 3 are important for systemin-mediated resistance to chewing M. sexta larvae and for Mi-1-mediated resistance to aphids, two feeding guilds which cause very different types of damage and induce distinct cellular responses (Li et al. 2006; Thompson and Goggin 2006). Some insects are known to induce direct and indirect defenses via fatty acid-amino acid conjugates (FACs) present in oral secretions or regurgitates (Alborn et al. 1997; Halitschke et al. 2003). It will be exciting to find out whether FACs employ the same signaling components as mechanical wounding. Stress-responsive MAPKs of the A and B1 groups seem to be major convergence points for many stress signals. Loss-of-function of these MAPKs has large

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consequences for defenses and protection against stress. Silencing of SIPK, WIPK and their tomato orthologs renders plants not only susceptible to pathogens, but also to chewing insects, aphids, and abiotic stress (Del Pozo et al. 2004; Ekengren et al. 2003; Kaitheri Kandoth et al. 2007; Li et al. 2006; Samuel and Ellis 2002; Seo et al. 2007; Sharma et al. 2003). On the other hand, gain-of-function studies revealed a more specific function, e.g., in the induction of the hypersensitive response (Ren et al. 2002; Yang et al. 2001; Zhang and Liu 2001). Since pathogen- and woundinduced signaling components often crosstalk, it is likely that overexpression of certain active MAPKs will not increase resistance to insects. However, this was never directly tested so far. Defense responses to wounding or herbivory and responses to pathogens are clearly distinct but both mediated by the same MAPKs. This raises a fundamental question. How is signaling fidelity and specificity generated via these highly promiscuous kinases? Moreover, what are the molecular mechanisms that lead to signal-specific cellular responses? This is a much debated problem in animal and yeast signal transduction (Pouyssegur and Lenormand 2003; Sabbagh et al. 2001). Explanations are based on the concept of a signaling network. In such a network, specific signaling components (nodes) are highly interconnected, respond to multiple input signals and relay the signal to various downstream nodes. MAPKs of the A-group represent focal nodes that respond to a high number of input signals. Positive and negative feedback-mechanisms among nodes, switch-like signaling behavior and signal amplification increase the complexity of the network. This complexity enables flexible responses and integration of multiple signals which is important for natural settings in which plants may be successively or synchronously challenged by multiple stressors. Several solutions have been proposed for the problem of signaling fidelity based on work with yeast or animal systems. There is some evidence that similar mechanisms may operate in plants; however, they are generally not well corroborated. MAPKs can be activated in either a prolonged or a transient manner. For specific mammalian MAPKs it has been established that the kinase activation kinetics determine the cellular response. Nerve growth factor induces prolonged activity of the mammalian MAPKs ERK1 and ERK2 and differentiation of PC12 cells into neuron-like cells. In contrast, epidermal growth factor induces transient ERK1/2 activation and PC12 cell proliferation, but not differentiation. The different activation kinetics were generated through differential interaction of growth factor receptors with interacting proteins which in turn resulted in assembly of stable or short-lived signaling complexes (Kao et al. 2001). Stress-responsive plant MAPKs are also known to exhibit different activation kinetics in response to different stimuli. It had been suggested that wounding-related signals lead to transient, and pathogen-related signals lead to prolonged MAPK activation (Higgins et al. 2007; Ren et al. 2002). The activation kinetics are likely to be a function of the activating signal, the nature of the ligand-receptor interaction, and transcriptional and postranslational activation of MAPKs and negative regulators such as protein phosphatases. In addition, negative and positive feedback loops affect activation kinetics. It had been shown that wound- and systemin-inducible MAPK cascades enter a refractory state after an initial activation indicating the


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presence of transient negative regulators of the cascade (Bögre et al. 1997; Holley et al. 2003; Yalamanchili and Stratmann 2002). Such a behavior indicates that sustained activity and inducibility of MAPKs is actively prevented, at least in response to wound-related signals. However, no mechanism has been revealed in plants that would explain how MAPK activation kinetics are interpreted by the signaling network to result in specific cellular responses. Interaction with other proteins may modulate the activity of MAPKs or determine signaling specificity. Well known examples from animals and yeast are scaffolding proteins which specifically interact with MAPKs (Breitkreutz and Tyers 2002; Morrison and Davis 2003; Pouyssegur and Lenormand 2003; Sabbagh et al. 2001). A specific MAPK cascade can be assembled by tethering a specific MAPK to the scaffolding protein which also interacts with specific MAPKKs and MAPKKKs. This would establish signaling fidelity by excluding competitor MAPKs. Since homologs of the yeast and animal scaffolding proteins are not present in plants, it is not known whether similar mechanisms are used in plants to combine cascade components. However, A MAPKKK from alfalfa had been shown to interact with a MAPK suggesting that the MAPKKK itself may function as a scaffold (Nakagami et al. 2004). Another way to generate specific responses via a signaling network is by differential activation and inactivation of multiple nodes that respond to multiple input signals. For example A-group MAPKs, ethylene, reactive oxygen species, Ca2+ , and proton fluxes participate in almost any stress signaling pathway. Each input signal has the potential to activate all these intermediate signals and signal transducers in an input-specific way via specific interactions with receptors that generate a specific pattern of network activity. A receptor-mediated input-specific signaling fingerprint is reflected in the kinetics and amplitude of the activity of individual nodes. This scenario can explain seemingly inconsistent MAPK gain- and loss-of-function experiments. Both approaches severely perturb the signaling network by overstimulation or disruption of a central node. Therefore, loss- and gain-of-function approaches might sometimes be more informative with regard to the rank of a node within the cellular signaling network than for the function of individual nodes in response to a particular input signal. It will be essential in the future to complement such studies with detailed biochemical analyses which address the dynamics of interactions among signaling nodes. Recent progress in phosphoproteomics (Peck 2006) and techniques used to study protein-protein interactions should reveal the dynamics of posttranslational modifications as well as protein complex assembly and disassembly in a signaling network in response to various input signals including herbivory. This would also involve the characterization of enzymatic parameters which are largely unknown for plant MAPKs, with only a few exceptions (Zhang and Klessig 1997). Such data were used to model the response of animal MAPK cascades which revealed that MAPK cascades function more like an on-off switch rather than in signal amplification (Ferrell 1996; Kolch et al. 2005). Compartmentalization and tissue-specific expression may further contribute to signaling specificity. To date, this has not been explored for most MAPKs; however, some MAPKs are known to be expressed in many different cell types (Ren et al. 2006). Upon activation, some MAPKs translocate from the cytosol

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into the nucleus to activate transcription (Ahlfors et al. 2004; Lee et al. 2004; Ligterink et al. 1997). Others stay in the cytosol to activate cytosolic substrates (Liu and Zhang 2004), still others are permanently present in the nucleus (Munnik et al. 1999), or translocate from the nucleus to the cytosol (Samaj et al. 2002). MAPKs are likely to exhibit a broad substrate specificity (Feilner et al. 2005), but a more specific MAPK-substrate interaction may be determined by compartmentspecific substrate availability. Unfortunately, only a few in vivo plant MAPK substrates have been identified so far. Protein arrays were developed to determine putative substrates for AtMPK3 and AtMPK6. Both MAPKs phosphorylated 48 and 39 functionally diverse proteins, respectively, and many of these substrates were phosphorylated by both MAPKs (Feilner et al. 2005). Similarly, Stulemeijer et al. (2007) carried out a PepChip Kinomics Slide Analysis and found that LeMPK1, 2, and 3 share many tripeptide phosphorylation motifs. In addition, each LeMPK phosphorylated specific substrates. This shows that the potential substrate range for plant MAPKs is very broad. It also implies that mechanisms which establish signaling fidelity through restricting substrate availability are important for fine tuning signaling networks in order to produce adequate output responses. A recent exciting development, based on the chromatin immunoprecipitation technique, is that yeast MAPKs may directly interact with transcribed regions of their target genes or associated histones and components of the transcriptional machinery (Chow and Davis 2006). An exciting future task will be to make use of the ever increasing amount of data on plant MAPKs and associated proteins to generate mathematical models of stress signaling networks. The predictive power of such models is increasingly being recognized for MAPK signaling in animal cells. Models revealed unexpected behavior of the signaling network that could not have been identified experimentally (reviewed in Kolch et al. 2005). Note added in proof: After submission of this article, an important paper was published by Wu et al. showing that FACs from M. sexta oral secretions activate SIPK and WIPK in N. attenuata. Silencing of these MAPKs revealed their critical role for FAC-induced JA-, SA-, and ethylene biosynthesis, and for the expression of a range of defense and signaling genes. (Wu J, Hettenhausen C, Meldau S, Baldwin IT (2007) Herbivory rapidly activates MAPK signaling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuata. Plant Cell 19:1096–1122).

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Chapter 17

Jasmonate Biosynthesis and Signaling for Induced Plant Defense against Herbivory Andreas Schaller and Annick Stintzi

Jasmonates are a growing class of signaling molecules and plant hormones which are derived from polyunsaturated fatty acids via the octadecanoid pathway, and characterized by a pentacyclic ring structure. Until recently, jasmonic acid has been viewed as the end product of the pathway and as the bioactive hormone. It becomes increasingly clear, however, that biological activity is not limited to jasmonic acid, but extends to, and may even differ between its many metabolites and conjugates as well as its biosynthetic precursors. Like other plant hormones, jasmonates exhibit a broad spectrum of physiological activities, ranging from seed germination, over reproductive development, all the way to senescence. Jasmonates also serve important roles as signaling molecules in plant defense, particularly defense against insect herbivores and necrotrophic patghogens. In this chapter, we will briefly discuss each step of the octadecanoid pathway, emphasizing on those that are relevant for the regulation of jasmonic acid biosynthesis, and on insights derived from the recently solved crystal structures of two of the pathway’s enzymes. With respect to jasmonate signaling, we will focus on their role as signal molecules in the systemic defense response against insect herbivores, and on jasmonate-dependent activation of defense gene expression.

17.1 Introduction Since the identification of methyl jasmonate (MeJA) as a secondary metabolite in essential oils of jasmine in 1962 (Demole et al. 1962), many related compounds have been discovered in a wide range of plants, and are collectively referred to as jasmonates. Jasmonates share their biosynthetic origin from oxygenated polyunsaturated fatty acids, and a substituted pentacyclic ring as a common structural element. In the early 1980s, their widespread occurrence throughout the plant kingdom (Meyer et al. 1984), and their growth-inhibitory (Dathe et al. 1981), and A. Stintzi University of Hohenheim, Institute of Plant Physiology and Biotechnology, D-70599 Stuttgart, Germany e-mail: [email protected] A. Schaller (ed.), Induced Plant Resistance to Herbivory, C Springer Science+Business Media B.V. 2008

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senescence-promoting activities (Ueda and Kato 1980) were established. First indications for a role of jasmonates in the regulation of gene expression were obtained by Parthier and co-workers who observed the accumulation of jasmonateinducible proteins (JIPs) in senescing barley leaves (Weidhase et al. 1987; MuellerUri et al. 1988). Seminal work of Farmer and Ryan subsequently demonstrated that MeJA and jasmonic acid (JA) induce the accumulation of proteinase inhibitors as a direct defense against insect herbivores (Farmer and Ryan 1990; Farmer et al. 1991). Shortly after, jasmonates were shown to mediate the elicitor-induced accumulation of antimicrobial phytoalexins in cell cultures (Gundlach et al. 1992), and the induction of vegetative storage proteins in soybean and Arabidopsis (Franceschi and Grimes 1991; Staswick et al. 1992). These findings greatly stimulated the interest in jasmonates as a new class of signaling molecules in plant defense against both insects and pathogens. In subsequent years, numerous mutants were characterized that are impaired in either jasmonate synthesis or response clearly establishing their function as plant defense regulators. Mutants impaired in jasmonate perception and signaling, including the coi1, jin1, and jai3 mutants in Arabidopsis (Feys et al. 1994; Berger et al. 1996; Lorenzo et al. 2004; Lorenzo and Solano 2005), coi1 in N. attenuata (Paschold et al. 2007), and the jai1 mutant in tomato (Li et al. 2004) fail to mount appropriate defense responses. Also compromised in the induction of defense responses are mutants that are deficient in the polyunsaturated fatty acid precursors of jasmonates (the fad3fad7fad8 and the spr2/LeFad7 mutants in Arabidopsis and tomato; McConn et al. 1997; Li et al. 2003), and mutants affected in the octadecanoid pathway for jasmonate biosynthesis, i.e. dad1, aos, acx1/5, jar1 in Arabidopsis (Park et al. 2002; Staswick et al. 2002; von Malek et al. 2002; Schilmiller et al. 2007), def1 in tomato (Howe et al. 1996) and jar4 in N. attenuata (Kang et al. 2006). In addition to their role as defense regulators, the characterization of biosynthesis and perception mutants established jasmonates as phytohormones in plant reproductive development. Many of the Arabidopsis mutants are male sterile due to defects in anther and pollen maturation (Feys et al. 1994; McConn and Browse 1996; Sanders et al. 2000; Stintzi and Browse 2000; Ishiguro et al. 2001; Park et al. 2002; von Malek et al. 2002; Schilmiller et al. 2007) while a defect in the maternal control of seed maturation appears to cause sterility in jai1 in tomato (Li et al. 2004). Other jasmonate-regulated developmental processes include root growth (Staswick et al. 1992), glandular trichome development (Li et al. 2004), tuber formation (Yoshihara et al. 1989; Pelacho and Mingo-Castel 1991), laticifer differentiation (Hao and Wu 2000), seed germination (Corbineau et al. 1988; Finch-Savage et al. 1996), carbon/nitrogen allocation (Creelman and Mullet 1997), senescence (Ueda and Kato 1980; Creelman and Mullet 1997), and tendril coiling (Falkenstein et al. 1991). A number of excellent review articles have recently been published on the biosynthesis of jasmonates and on their activity, particularly their contribution to the regulation of plant defense responses (Blee 2002; Liechti and Farmer 2002; Halitschke and Baldwin 2004; Howe 2004; Pozo et al. 2004; Schaller et al. 2004; Browse 2005; Lorenzo and Solano 2005; Schilmiller and Howe 2005; Delker

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et al. 2006; Wasternack 2006; Cheong and Choi 2007; Wasternack 2007), and we refer the reader to these articles for a comprehensive discussion of the available literature. In this chapter, we will emphasize on the most recent findings with respect to jasmonate biosynthesis, and discuss those aspects of jasmonate signaling that are relevant for the systemic induction of herbivore defenses and defense gene activation.

17.2 Jasmonate Biosynthesis The synthesis of jasmonates and many other oxylipins is initiated by lipoxygenases (LOXs), which catalyze the regio- and stereoselective dioxygenation of polyunsaturated fatty acids (reviewed by Blee 2002; Feussner and Wasternack 2002; Howe and Schilmiller 2002; Schaller et al. 2004; Wasternack 2007). Linoleic acid (18:2) and linolenic acid (18:3) are oxygenated by specific LOXs at C9 or C13 to result in the corresponding (9S)- or (13S)-hydroperoxy-octadecadi(tri)enoic acids, which feed into at least seven alternative pathways resulting in the formation of a large variety of oxylipins (Blee 2002; Feussner and Wasternack 2002). The first committed step in the two parallel pathways for JA biosynthesis (Fig. 17.1), i.e. the octadecanoid pathway from 18:3 and the hexadecanoid pathway from 16:3 (Weber et al. 1997), is performed by allene oxide synthase (AOS), an unusual cytochrome P450 which uses its hydroperoxide substrate as source for reducing equivalents and as oxygen donor, and is thus independent of molecular oxygen and NAD(P)H. AOS catalyzes the dehydration of 13(S)-hydroperoxy-octadecatrienoic acid (13-HPOT) to form an unstable allene oxide, 12,13(S)-epoxy-octadecatrienoic acid (12,13-EOT). In aqueous media, 12,13-EOT rapidly decomposes to ␣- and ␥-ketols, or undergoes cyclization to form 12-oxo-phytodienoic acid (OPDA). As opposed to spontaneous cyclization which results in a racemic mixture of OPDA enantiomers, allene oxide cyclase (AOC) ensures the formation of the optically pure 9S,13S enantiomer. Dinor-OPDA (dnOPDA) is generated in the parallel pathway from 16:3 (Fig. 17.1). The short half-life of 12,13-EOT in aqueous media (26 s at 0◦ C and pH 6.7; Hamberg and Fahlstadius 1990; Ziegler et al. 1999) and the optical purity of endogenous OPDA (Laudert et al. 1997) suggest tight coupling of the AOS and AOC reactions in vivo. However, physical contact of AOS and AOC in an enzyme complex does not seem to be required for stereochemical control of the cyclization reaction (Zerbe et al. 2007). Only 9S,13S-OPDA, i.e. one out of four possible OPDA stereoisomers, is a percursor for biologically active JA. AOC is thus crucially important to establish the enantiomeric structure of the cyclopentenone ring. The crystal structure of Arabidopsis AOC2 has recently been solved shedding light on how the enzyme exerts stereochemical control on the cyclization reaction (Hofmann et al. 2006). Considering the fact that cyclization occurs spontaneously in aqueous solution, AOC2 does not need to be much of a catalyst in terms of lowering the activation energy barrier. Indeed, binding of the substrate or the transition state does not involve any induced fit mechanism. The hydrophobic protein environment and very few ionic interactions with a glutamate residue (Glu23) and a tightly bound water molecule,

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Fig. 17.1 Biosynthesis of jasmonic acid. Polyunsaturated fatty acids (18:3 and 16:3) are precursors for jasmonic acid biosynthesis via the octa- and hexadecanoid pathways, respectively. The first three steps are localized in plastids and lead to the formation of 12-oxophytodienoic acid (OPDA) and dinor 12-oxophytodienoic acid (dnOPDA). The subsequent steps, including the reduction of OPDA (dnOPDA) to OPC8:0 (OPC6:0) followed by three (two) cycles of ␤-oxidation result in the production of jasmonic acid in peroxisomes. Arrows and broken arrows are used to differentiate between well characterized reactions and those steps that are still hypothetical and for which the corresponding enzymes remain to be identified. Further detail is given in the text

ensure binding and correct positioning of the substrate 12,13-EOT. Steric restrictions imposed by the protein environment enforce the necessary conformational changes of the substrate’s hydrocarbon tail resulting in the absolute stereoselectivity of the AOC2-mediated as opposed to the chemical cyclization reaction (Hofmann et al. 2006).


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The passive role of AOC2 in the steroselective synthesis of 9S13S-OPDA is reminiscent of dirigent proteins and the way in which they promote stereoselective formation of lignans. Lignan formation involves the regio- and sterochemically controlled oxidative coupling of two phenols, e.g. the formation of (+)-pinoresinol from two molecules of coniferylalcohol (Davin et al. 1997). In absence of dirigent proteins, resonance-stabilized phenoxy radical intermediates couple randomly to form a mixture of racemic lignans. Dirigent proteins, which lack a catalytic center, are believed to bind and orient the free radical intermediates allowing stereoselective coupling to occur (Davin et al. 1997). Interestingly, both dirigent proteins and AOC2 are distant members of the lipocalin family (Charron et al. 2005; Hofmann et al. 2006; Pleiss and Schaller unpublished observation). Lipocalins are ␤-barrel proteins comprising a central hydrophobic cavity for binding of small lipophilic molecules like steroids, pheromones, odorants, or retinoids. In fact most lipocalins do not function as enzymes but rather as binding proteins in olfaction, pheromone transport, retinol transport, and invertebrate cryptic coloration (Charron et al. 2005). Likewise, dirigent proteins and AOC2 act as binding proteins of the unstable lipophilic precursors (phenoxy radicals and allene oxides, respectively) to exert stereochemical control in the synthesis of lignans and OPDA. The formation of 9S,13S-OPDA as the first member of the jasmonate family with signaling activity concludes the plastid-localized part of the octa(hexa)decanoid pathway. Little is known about how OPDA (and/or dnOPDA) is transferred from plastids to peroxisomes, where the final steps of JA biosynthesis occur. Recently, the ABC transporter COMATOSE (CTS, also known as PXA1 (Zolman et al. 2001) or PED3 (Hayashi et al. 2002)) was shown to be involved in this process (Theodoulou et al. 2005). CTS catalyzes the ATP-dependent import of fatty acids into peroxisomes as substrates for ␤-oxidation. The cts mutant has reduced levels of JA, is impaired in wound-induced JA accumulation, and expresses the JA-dependent VSP1 gene at a lower level suggesting that CTS delivers substrates for JA synthesis into the peroxisomes, most likely (dn)OPDA, or the corresponding CoA esters (Theodoulou et al. 2005). However, other pathways for (dn)OPDA import must exist, as indicated by the residual levels of JA, and the lack of JA-deficiency symptoms (e.g., male sterility) in the cts mutant. Additional pathways for fatty acid import into peroxisomes, by diffusion or by an as yet unidentified mechanism, were also suggested by Fulda et al. (2004). Because of the higher pH in peroxisomes as compared to the cytoplasm, weak acids are predicted to be trapped in peroxisomes as the anion, and this may account for some of the (dn)OPDA import (Theodoulou et al. 2005). Once within the peroxisomes, 9S,13S-OPDA is reduced by 12-oxophytodienoate reductase (OPR3) to yield 3-oxo-2-(2’(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0), and dnOPDA is reduced to the corresponding hexanoic acid derivative (OPC-6:0; Schaller et al. 2000; Stintzi and Browse 2000; Fig. 17.1). The signaling properties of jasmonates bearing a cyclopentenone ring (OPDA, dnOPDA) are clearly distinct from JA and its derivatives which are characterized by the reduced cyclopentanone ring (Blechert et al. 1999; Stintzi et al. 2001; Taki et al. 2005). OPR3 may therefore be particularly important for the regulation of the relative levels of these two classes of signaling molecules.

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Even though OPR3 belongs to a small family of related flavin-dependent oxidoreductases (at least three in tomato, six genes in Arabidopsis, six in pea, eight in maize, ten in rice) it is functionally unique, as evident from JA-deficiency symptoms (male-sterility) of the OPR3 loss-of-function mutant in Arabidopsis (Sanders et al. 2000; Stintzi and Browse 2000) and tomato (Schaller and Stintzi unpublished). The genetic evidence is consistent with biochemical and structural data which suggest that all OPRs catalyze the reduction of ␣,␤-unsaturated carbonyls (conjugated enones) while only OPR3s and the rice ortholog OsOPR7 (Tani et al. 2007) are capable of reducing the 9S,13S enantiomer of OPDA (Schaller et al. 2000; Breithaupt et al. 2001, 2006; Strassner et al. 2002). Therefore, these are the only OPRs contributing to JA biosynthesis. The OPR1 isoforms from Arabidopsis, tomato, and rice, on the other hand, were shown to reduce numerous conjugated enones including 9R,13R-OPDA, but they do not accept 9S,13S-OPDA the precursor of biologically active JA (Schaller and Weiler 1997; Straßner et al. 1999; Schaller et al. 2000; Strassner et al. 2002; Sobajima et al. 2003; Tani et al. 2007). Recent crystal structure analyses provided insights into the mechanisms of substrate reduction and the remarkable differences in stereospecificity between OPR1 and 3 (Breithaupt et al. 2001, 2006; Fox et al. 2005; Malone et al. 2005). Consistent with the reaction mechanism proposed for the related Old Yellow Enzyme from yeast, the carbonyl moiety of the substrate forms hydrogen bonds with two histidine residues (His187 and His197 in tomato OPR1) which leads to a polarization of the ␣,␤-double bond. Consequently, hydride transfer from the reduced flavin cofactor to the substrate C␤ is facilitated, followed by a protonation of the C␣ by a tyrosine residue (Breithaupt et al. 2001). A comparison of the OPR1 and OPR3 structures revealed a more open active site cavity in OPR3, explaining its more relaxed specificity allowing reduction of both the (9S,13S) and (9R,13R) isomers of OPDA. Two residues in OPR1, Tyr78 and Tyr246 seem to act as gatekeepers, narrowing the active site and blocking the entry of 9S,13S-OPDA (Breithaupt et al. 2001, 2006). The shortening of the hexanoic and octanoic acid side chains in OPC-6:0 and OPC-8:0 to yield JA involves two or three rounds of ␤-oxidation, respectively. Prior to entry into the ␤-oxidation cycle, the carboxylic moiety needs to be activated as CoA ester. Co-expression with genes known to be involved in JA biosynthesis suggested At1g20510, a member of the ATP-dependent acyl-activating family of enzymes, as a candidate for the required Acyl-CoA ligase. The recombinant enzyme was found to activate OPDA and OPC-8:0 in vitro, as well as mediumto-long straight-chain fatty acids (Koo et al. 2006). Loss-of-function mutants in Arabidopsis accumulated less JA in response to mechanical wounding providing direct evidence for a role in JA biosynthesis. Hyper accumulation of OPC-8:0 in the null mutants supported OPC-8:0 as the physiological substrate, and the enzyme was thus named OPC-8:0 CoA Ligase1 (OPCL1; Koo et al. 2006; Fig 17.1). A closely related enzyme (At4g05160) was suggested by Schneider et al. (2005) as a candidate OPC-6:0-CoA ligase. Consistent with such a role, the enzyme is located in peroxisomes and its expression is induced by MeJA. However, the recombinant enzyme was found to prefer medium-chain fatty acids over OPC-6:0 as substrates in vitro (Schneider et al. 2005), and the corresponding loss-of-function mutant exhibited


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wild-type levels of JA (Koo et al. 2006). Its contribution to JA biosynthesis is thus questionable. Beta-oxidation itself involves three core enzymes, acyl-CoA oxidase (ACX), multifunctional protein (MFP; comprising enoyl-CoA hydratase and ␤-hydroxyacyl-CoA dehydrogenase activities), and 3-ketoacyl-CoA thiolase (Fig. 17.1). Despite early findings implicating ␤-oxidation in JA biosynthesis (Vick and Zimmerman 1984), direct evidence for the contribution of these enzymes is very recent. ACX1A was shown to catalyze the first step in the ␤-oxidation of OPC-8:0-CoA, and was found to be responsible for the bulk of wound-induced JA production in tomato. Consistant with its essential role in JA biosynthesis, the acx1 tomato mutant was impaired in wound-induced defense gene activation and insect resistance (Li et al. 2005). In Arabidopsis, ACX1 is responsible for about 80% of JA production after wounding (Cruz Castillo et al. 2004; Schilmiller et al. 2007), and only the acx1/5 double mutant showed hallmarks of severe JA deficiency, including impaired insect resistance and reduced male reproductive function (Schilmiller et al. 2007). Also the aim1 mutant in Arabidopsis, which is disrupted in one of two MFP genes, is impaired in wound-induced accumulation of JA and expression of JA-dependent genes (Delker et al. 2007). Among the five 3-ketoacyl-CoA thiolase genes in Arabidopsis, KAT2 appears to be the one most relevant for JA biosynthesis. In transgenic plants silenced for KAT2 expression, wound-induced JA accumulation was markedly reduced (65%–80%), and the induction of a JA-dependent marker gene (JR2) impaired, both locally at the site of wounding and systemically (Cruz Castillo et al. 2004). As the final step in JA biosynthesis, the JA-CoA ester has to be hydrolyzed to release the free acid. Candidate acyl-thioesterases have been identified in Arabidopsis, two of which appear to be peroxisomal (AtACH1 and AtACH2; Tilton et al. 2000), but a direct involvement in JA biosynthesis remains to be shown. Traditionally, JA has been viewed as the end product of the pathway and the bioactive hormone. The many different metabolites of JA and the enzymatic steps in their interconversion were thought to contribute to hormone homeostasis to sustain and control the levels of active JA. This view is changing, however, and E.E. Farmer takes the extreme position to consider JA as a mere precursor of bioactive hormones, i.e. JA conjugates (Farmer 2007). Indeed, there is clear evidence showing that some JA metabolites have unique signaling properties, and that some processes are not controlled by JA but rather by JA derivatives (reviewed by Wasternack 2007). For example, the nyctinastic leaf movement in Albizzia depends on a specific enantiomer of 12-OH-JA-O-glucoside (Nakamura et al. 2006a, b), 12-OH-JA (tuberonic acid) has long been implicated in potato tuber formation (Yoshihara et al. 1989), and in defense signaling, the active signal appears to be the amide-linked isoleucine conjugate JA-Ile, rather than JA itself (Staswick and Tiryaki 2004; Kang et al. 2006). The relevance of JA-Ile as a signal in its own right was first demonstrated by Wasternack and co-workers who observed that JA and JA-Ile induce different responses when applied exogenously to barley leaves, and that they are both active without being inter-converted (Kramell et al. 1997; Wasternack et al. 1998). Biosynthesis of JA-Ile involves the adenylation of JA, followed by the exchange of

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AMP with isoleucine, and is catalyzed by the amino acid conjugate synthetase JAR1 (Staswick et al. 2002; Staswick and Tiryaki 2004). The Arabidopsis jar1 mutant is insensitive to exogenously applied JA with respect to root growth inhibition, induced resistance to Phytium irregulare, induced systemic resistance (ISR), and protection against ozone damage (Staswick and Tiryaki 2004, and references therein). Furthermore, silencing of the JAR1 ortholog in N. attenuata compromised defense gene induction and resistance against M. sexta (Kang et al. 2006). Therefore, conjugation to Ile appears to be necessary for at least a subset of the jasmonate-regulated processes. Further support for the specific signaling function of JA-Ile is provided by the recent observation that unlike JA or OPDA, JA-Ile mediates the specific degradation of repressors of jasmonate-dependent gene expression (Thines et al. 2007, see below).

17.3 Jasmonate Signaling in the Systemic Induction of Herbivore Defenses The systemic induction of defensive proteinase inhibitors in tomato plants in response to local wounding or herbivore attack was discovered by Ryan and coworkers 35 years ago and has since served as a model system to study long-range signaling processes in plants (Green and Ryan 1972). A number of chemical signals were identified in the Ryan lab that are intricately involved in the systemic induction of defense responses. This includes systemin peptides which are derived from larger precursor proteins by proteolytic processing (Pearce et al. 1991; McGurl et al. 1992; Ryan and Pearce 2003; Narváez-Vásquez and Orozco-Cárdenas this volume), oligogalacturonides which are generated by a polygalacturonase systemically induced after wounding (Bergey et al. 1999), and jasmonates which are derived from the octadecanoid pathway (Farmer and Ryan 1990; Farmer et al. 1991). Farmer and Ryan proposed a model according to which primary wound signals like oligogalacturonides and systemin trigger the activation of the octadecanoid pathway resulting in a burst of JA production which ultimately leads to the activation of defense genes (Farmer and Ryan 1992). The wound signaling pathway, its interaction with other hormone and defense signaling pathways, and the role of individual signaling molecules were subject of numerous recent reviews (Pieterse et al. 2006; Ryan and Pearce 2003; Stratmann 2003; Howe 2004; Lorenzo and Solano 2005; Schilmiller and Howe 2005; Wasternack et al. 2006; Cheong and Choi 2007), and systemic wound signaling is discussed in detail by Schaller and Howe (this volume). Here we will focus on the specific role of jasmonates in the activation of defense gene expression. Since the discovery of the systemic wound response researchers have been fascinated by the question of what the long-distance signal might be (Ryan 1992; Bowles 1998). The search for the systemic wound signal in tomato plants led to the discovery of systemin in the Ryan lab, and its role as the systemically mobile signal was supported by numerous observations: Systemin was shown to be


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necessary and sufficient for the systemic wound response, it triggers the synthesis and accumulation of JA, it is mobile within the phloem when applied to wound sites, and its precursor protein is specifically expressed in the vasculature. Based on these findings, a model had been proposed, according to which systemin is proteolytically released from its precursor upon wounding, loaded into sieve elements, and delivered along source-to-sink gradients into systemic tissues where it stimulates the synthesis of jasmonic acid as a secondary signal for the induction of defense gene expression (Ryan 2000). However, this model is not entirely consistent with recent findings from the Howe lab, demonstrating that systemic signaling requires the activity of systemin and the synthesis of JA only in the wounded, not in the systemic leaves. Furthermore, the induction of defense genes in systemic tissues depends on JA perception and signaling, but not on the capacity to synthesize JA (Li et al. 2002, 2003; Schaller and Howe this volume). These findings are in complete agreement with the observation that expression of octadecanoid pathway genes is induced by wounding at the site of tissue damage but not systemically, and that there is a dramatic increase in JA levels in wounded leaves but not in unwounded tissues (Strassner et al. 2002). The data suggest that systemin acts at the site of tissue damage, to strengthen the systemic wound response by boosting the octadecanoid pathway, for the generation of the long-distance signal, maybe JA itself or one of its derivatives (Ryan and Moura 2002; Stratmann 2003; Schilmiller and Howe 2005; Schaller and Howe this volume). The joint role of (pro)systemin and the octadecanoid pathway in the generation of a phloem-mobile signal for systemic induction of defense genes is consistent with their localization in the vasculature of tomato plants. Several of the octadecanoid pathway enzymes – LOX, AOS, and AOC – are located in the companion cellsieve element complex (Hause et al. 2000, 2003a), whereas prosystemin accumulates in cells of the phloem parenchyma (Jacinto et al. 1997; Narváez-Vásquez and Ryan 2004; Narváez-Vásquez and Orozco-Cárdenas this volume). The presence of prosystemin and octadecanoid pathway enzymes in different cell types of the vascular bundle suggests a model in which systemin is released from phloem parenchyma cells in response to wounding, is then perceived at the cell surface of neighboring companion cells, where it triggers the activation of the octadecanoid pathway for the production of the systemic signal, and its release into the sieve elements for long-distance transport (Schilmiller and Howe 2005; Wasternack 2006). The onset of JA accumulation at the site of tissue damage is almost instantaneous and peaks at around 1 hour after wounding (Doares et al. 1995; McConn et al. 1997; Ziegler et al. 2001; Strassner et al. 2002), and must therefore be independent from changes in gene expression. How the initial burst in jasmonate production is controlled, and how the production of jasmonates is limited in unstressed tissues are still open questions. It is obviously not the level of octadecanoid pathway enzymes, which are constitutively expressed and abundant in unstressed tomato leaves (Hause et al. 2003b; Stenzel et al. 2003b; Li et al. 2004). These enzymes do either not have access to their respective substrates, in this case substrate availability would be limiting for jasmonate production, and/or constitutively expressed octadecanoid pathway

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enzymes may be inactive and require posttranslational modification for activation. Both hypotheses are consistent with the repeated observation that transgenic plants overexpressing octadecanoid pathway enzymes do not have elevated resting levels of JA, but show increased JA production after wounding (Wang et al. 1999; Laudert et al. 2000; Park et al. 2002; Stenzel et al. 2003a). Primary substrates for the octa(hexa)decanoid pathway are polyunsaturated fatty acids (18:3, 16:3) which are abundant in chloroplast lipids, but not readily available as precursors for JA production in unwounded plants. Additional lipid-bound substrates may include fatty acid hydroperoxides, OPDA, and dnOPDA generated by LOX, AOS, and AOC from esterified rather than free fatty acids (Fig. 17.1; Buseman et al. 2006). The availability of these substrates as precursors for JA biosynthesis is controlled by lipolytic activities, which may include different types of phospholipases and acyl hydrolases (reviewed by Delker et al. 2006). However, in contrast to DAD1 which is required for JA production during male reproductive development (Ishiguro et al. 2001), the lipase(s) involved in the wound-induced burst of JA production have not been identified at the molecular level. An interesting scenario of how post-translational modification may contribute to the regulation of octadecanoid pathway activity and the initial burst of JA production after wounding has recently been derived from the crystal structure of tomato OPR3. The enzyme was found to crystallize as a homodimer, in which each protomer blocks the active site of the other (Breithaupt et al. 2006). Dimerization of OPR3 concomitant with a loss of activity was also observed in solution, suggesting that OPR3 activity may be controlled in vivo by regulation of the monomer/dimer equilibrium. The crystallized OPR3 dimer was found to be stabilized by a sulfate ion at the dimer interface. Intriguingly, the sulfate is located close to Tyr364 and is positioned perfectly to mimic a phosphorylated tyrosine residue suggesting that dimerization and hence OPR3 activity may be regulated by phosphorylation of Tyr364 in vivo (Breithaupt et al. 2006). This scenario would be consistent with a proposed role for reversible protein phosphorylation in JA biosynthesis and the regulation of the wound response (Rojo et al. 1998; Schaller and Oecking 1999; Stratmann this volume). Following the production of JA at the site of tissue damage and transmission of the long distance signal, the activation of defense responses in systemic tissues requires COI1 (Li et al. 2002, 2003). COI1 was identified many years ago as an essential component of the JA signaling pathway in a screen for insensitivity to coronatine, a structural analogue of JA-Ile (Feys et al. 1994; Xie et al. 1998). The finding that COI1 is part of the Skp/Cullin/Fbox (SCF)COI1 complex, a type of E3 ubiquitin ligase, suggested that JA signaling is controlled by a negative regulator that is ubiquinated specifically by SCFCOI1 , targeted for destruction by the 26S proteasome, resulting in the activation of JA responses (Xu et al. 2002). The negative regulators of JA signaling have remained elusive until, very recently, two independent studies led to the identification of JAZ (jasmonate ZIM domain) proteins as targets of SCFCOI1 (Chini et al. 2007; Thines et al. 2007). JAI3 was identified in Arabidopsis as a member of the JAZ protein family which negatively regulates MYC2, the key transcriptional activator of JA-dependent genes


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(Lorenzo et al. 2004; Chini et al. 2007). In the jai3-1 mutant, a splicing acceptor site mutation results in the formation of a truncated JAI3 (JAZ3) protein, which is no longer subject to COI1-dependent degradation resulting in a JA-insensitive phenotype (Chini et al. 2007). Thines et al. (2007) identified the JAZ family in a search for transcripts rapidly upregulated by JA. JAZ1 was shown to be a negative regulator of JA-dependent genes, which is degraded in a COI1-dependent manner in response to JA. They further demonstrated that the physical interaction of COI1 and JAZ1 is promoted by JA-Ile, but not JA, MeJA, or OPDA. This interaction was independent of other protein factors suggesting that the COI1-JAZ1 complex is the site of JA-Ile perception (Thines et al. 2007). These exciting findings explain beautifully how jasmonate signals may activate defense responses in systemic tissues by triggering the COI1-dependent degradation of JAZ proteins, thus relieving MYC transcriptions factors from negative regulation, resulting in the transcriptional activation of defense genes.

17.4 Perspectives Despite tremendous progress in recent years, many open questions remain with respect to jasmonate biosynthesis and signaling. As far as JA biosynthesis is concerned, surprisingly little is known of how it all starts. The question is not as trivial as it may seem. Obviously, plastid-localized 13-LOXs catalyze the formation of fatty acid hydroperoxides as the first step in JA biosynthesis, but LOX substrates may include free polyunsaturated fatty acids liberated from chloroplast lipids by unidentified lipases (Bachmann et al. 2002), or fatty acids that remain esterified in membrane lipids (Brash et al. 1987). Indeed, octa(hexa)decanoid pathway intermediates are present in complex lipids in Arabidopsis. Seventeen complex lipids have been identified already, including mono- and digalactosyldiacylglycerols (MGDGs and DGDGs), as well as phosphatidylglycerol, with OPDA and dnOPDA esterified in the sn1 and/or sn2 positions (Stelmach et al. 2001; Hisamatsu et al. 2003, 2005; Buseman et al. 2006). The very rapid (within 15 min) and very large (200–1000 fold) increase of complex lipids containing two oxylipin chains after wounding, as well as the relative abundance and positional specificity of OPDA and dnOPDA in the different galactolipid species which reflects the composition of galactolipids prior to wounding, suggest that they were generated in situ by direct conversion of esterified 18:3 and 16:3 (Buseman et al. 2006). Consistent with this notion, oxylipin-containing galactolipids and AOS are co-localized in the thylakoid membrane fraction of Arabidopsis chloroplasts (Böttcher and Weiler 2007). In the light of such a diversity of oxylipin-containing lipids in Arabidopsis, the identification of the lipases that are involved in their release, and of the mechanisms in control of their activities are urgent problems to be addressed in the future. A long-standing question with respect to jasmonate signaling concerns the activities of different jasmonate family members. The traditional view of JA being the bioactive hormone was challenged by Seo et al. who suggested that defense signaling

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relies on the formation of MeJA by a JA carboxyl methyltransferase (Seo et al. 2001). Furthermore, the JAR1-dependent conjugation to Ile was found to be required for (at least a subset of) JA responses (Staswick and Tiryaki 2004). The identification of the COI1-JAZ1 complex as the perception site for JA-Ile but not for JA, MeJA, or OPDA provided strong support for the importance of JA-Ile as a regulator of jasmonate response genes (Thines et al. 2007). However, this finding does not preclude the possibility that jasmonates other than JA-Ile may control subsets of COI1-dependent genes, as it was shown for OPDA (Stintzi et al. 2001). Conceivably, different members of the JAZ protein family may interact with different transcription factors, and thus, may each regulate part of the response to jasmonates. The degradation of these JAZ proteins and, consequently, transcriptional activation depend on their interaction with COI1, which in case of JAZ1 is stimulated by JA-Ile. Other jasmonates may promote the interaction of COI1 with different JAZ repressors, target them for degradation, and release inhibition of jasmonate-responsive genes. Despite the progress made in recent years, there are still many open questions and discoveries waiting to be made concerning both, jasmonate biosynthesis and signaling, but also related to the transport of jasmonates and communication between organelles, plant organs, and organisms. Acknowledgments The authors thank the German Research Foundation (DFG) and the German Academic Exchange Service (DAAD) for support.

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Part B

Signals Between Plants and Insects

Chapter 18

Caterpillar Secretions and Induced Plant Responses Gary W. Felton

Saliva and the glands that produce it could be the leading edge of rapid evolutionary adaptation to new food sources. –(Tabak and Kuska 2004)

Plants employ both direct inducible defenses (those that directly affect the physiology of the herbivore) and indirect induced defenses (defenses that attract or enhance the effectiveness of natural enemies of herbivores). During the last two decades, there has been a proliferation of studies showing that the oral secretions and saliva of herbivores play important roles in mediating inducible responses. Much of the study has centered on lepidopteran larvae (i.e., moths and butterflies) – no doubt due to their economic importance as agricultural pests, but also due to the relative ease of working with their secretions. In this chapter, I will not attempt a comprehensive review on the subject, but instead will focus this discussion on the secretions of larval Lepidoptera.

18.1 Introduction Caterpillars feed by chewing and ingesting plant tissue, resulting in tissue damage which is sufficient to trigger plant defense responses. Investigators have attempted to mimic their feeding style by wounding plant tissues with scissors, tracing wheels, cork borers, or most elegantly with the MecWorm (Mithöfer et al. 2005). While each of these treatments causes wound responses in the plant, both locally at the site of wounding and systemically in unwounded tissues, it has become clear that wounding by itself cannot account for the full breadth and the specificity of plant responses to herbivory. Therefore, herbivore-associated factors have to exist which modulate the wound response and/or elicit additional reactions in the host plant (Hartley and Lawton 1987; Turlings et al. 1990; Mattiacci et al. 1995; Korth and Dixon 1997; Baldwin et al. 2001; Gomez et al. 2005). Insect-derived elicitors may be present in the faeces and, most prominently, in oral secretions. G.W. Felton Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA e-mail: [email protected] A. Schaller (ed.), Induced Plant Resistance to Herbivory, C Springer Science+Business Media B.V. 2008

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To determine if oral secretions play any additional role in the specificity of the plant wound responses, the secretions are typically applied to artificially wounded plants. The diminutive size of insect herbivores other than caterpillars, such as aphids, whiteflies, thrips, etc. makes their study more intractable. The piercing-sucking feeding style of the aphids, leafhoppers and other insects adds another level of difficulty when trying to mimic their feeding. The majority of the research on their saliva has focused on its roles in detoxifying plant compounds (Miles 1999) and in transmission of plant pathogens (Fereres 2007), and less on its role in induction of plant defenses (Williams et al. 2005). Despite their small size, saliva can be collected from aphids and whiteflies using a feeding membrane (Miles and Harrewijn 1991). The saliva of insect herbivores was the subject of a review by my laboratory (Felton and Eichenseer 1999); however, the review is now over 8 years old and substantial progress has occurred since. Herein, this chapter focuses on recent advances on caterpillar secretions.

18.2 Oral Secretions and Saliva – A Clarification Larval Lepidoptera possess two pairs of salivary glands: the labial glands and mandibular glands (Bordas 1903; Parthasarathy and Gopinathan 2005). The silk secretions of the labial glands of the silkworm Bombyx mori have been known and used for thousands of years for clothing (Yokoyama 1963) and medicine (Lev 2003), yet the fact that the same glands can produce watery saliva is not widely appreciated by many entomologists. I will define saliva sensu stricto, to refer only to the secretions of the labial and mandibular salivary glands. The labial glands are generally long and tubular, and converge into a common duct and then are released from the spinneret (Fig. 18.1); the same structure from where silk is released. In the case of the noctuid Helicoverpa (=Heliothis) zea, the larval labial glands were mistakenly believed to empty and secrete into the digestive tract at the pharynx (Chauthan and Callahan 1967), although this has been corrected by several authors (Standlea and Yonke 1968; Macgown and Sikorowski 1982; Felton and Eichenseer 1999). The mandibular glands are tubular but highly variable among species (Parthasarathy and Gopinathan 2005). Frequently they are relatively minute and restricted to the head region (Eichenseer et al. 2002); in other cases they may be nearly as long as the labial glands and extend well into the thorax (Vegliante 2005). The glands open into the lumen of the mandibular adductor apodeme. In some species, a second lobed accessory gland may be found attached to the mandibles, which open through numerous pores on both sides of the mandibles (Vegliante 2005). The chemical characterization of caterpillar saliva has been barely touched. Proteins are present in labial gland saliva (Liu et al. 2004), but other macromolecules and small molecules have not been reported. Mandibular gland secretions may also contain proteins (Eichenseer et al. 1999), but these secretions have long been noted to be rich in different classes of lipids, such as sterols and triglycerides (Wroniszewska 1966; Mossadegh 1978; Eichenseer et al. 2002; Howard and Baker 2004).

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Fig. 18.1 Electron micrograph of frontal view of head of larval Helicoverpa zea. Arrow points to spinneret, the external secretory structure for silk and labial gland saliva. Aperture in the spinneret for the release of silk and/or saliva is clearly visible. (Micrograph courtesy of Michelle Peiffer)

The salivary secretions of the labial and mandibular glands are distinct from the regurgitant or ‘oral secretions’ (=OS) which have been widely used in studies of plant-herbivore interactions (Felton and Eichenseer 1999; Liu et al. 2004). Multiple laboratories [e.g., Baldwin (Roda et al. 2004), Boland (Arimura et al. 2005), De Moraes (De Moraes and Mescher 2004), Dicke (Mattiacci et al. 1995) Korth (Korth and Dixon 1997), Mori (Yoshinaga et al. 2006), Pare (Truitt and Pare 2004), Schmelz (Schmelz et al. 2006), Tumlinson (Tumlinson and Lait 2005), Turlings (Gouinguene et al. 2003) and Wang (Yan et al. 2005)] are working on OS. These secretions arise from the herbivore’s digestive system (von Dahl et al. 2006) and may contain insect-derived substances (e.g., digestive enzymes), as well as plant material in various stages of digestion. Because the OS are collected as regurgitant, it is entirely possible that small amounts of labial and mandibular saliva are collected during this process. The active components of caterpillar OS have most often been shown to be fatty acid-amino acid conjugates (=FACs; Pohnert et al. 1999a, b; Halitschke et al. 2001; Lait et al. 2003; Spiteller and Boland 2003a, b; Yoshinaga et al. 2005; Aboshi et al. 2007) including volicitin, the first FAC identified as an elicitor (Alborn et al. 1997). More recently, plant protein fragments of chloroplastic ATP synthase ␥-subunit protein (termed inceptin) found in the OS of the fall armyworm were shown to induce indirect and direct defenses in cowpea (Schmelz et al. 2006, 2007). Also, OS may contain microbes ingested with food or derived from resident microbial populations in the digestive system (Spiteller et al. 2000; Cardoza et al. 2006).

18.3 Experimental Approaches for Studying Oral Secretions and Saliva The function of saliva in caterpillars can be assessed using surgical, pharmacological, and/or molecular approaches. Surgical techniques have been applied to prevent caterpillars from secreting saliva. These techniques involve the extirpation of the

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salivary glands or cauterization of the spinneret to prevent salivation. Wroniszewska (1966) surgically removed the mandibular glands of Galleria mellonella and demonstrated that the loss of the glands causes a considerable decrease in the growth rate of larvae, although the specific cause for slower growth rates was not noted. Similarly Mossadegh (1978) surgically removed mandibular glands to examine the role of their secretions on silk filaments, and also cauterized the spinnerets (using a heated needle) to prevent silk deposition. Cauterization of the spinneret has also been used to examine the role of silk in trail following behavior in social caterpillar species (Fitzgerald 1993, 2003). Cauterization of the spinneret and surgical removal of the labial glands have been used to examine the function of labial saliva (Musser et al. 2002, 2005a, b, 2006; Bede et al. 2006) We have recently used a medical cauterization tool which more consistently accomplishes a uniform cauterization (Peiffer and Felton 2005). Machida (1965) removed the Lyonet’s gland (Filippi’s gland) or severed its duct to determine the impact of the gland on silk secretion. Surgical removal of salivary glands, particularly the mandibular glands that are often relatively small, requires a high degree of skill to avoid killing larvae. Furthermore, the interpretation of results using surgically invasive procedures should be cautioned because rupture of the cuticle causes a massive cascade of wound responses (Jarosz 1993; Han et al. 1999). Cauterization of the spinneret avoids this invasive procedure, but is limited to examining labial gland secretions. In some species (e.g., Pieris rapae) cauterization may adversely affect larval feeding behavior (Musser et al. 2006). Despite the robust size of Manduca sexta larvae, their spinnerets are comparatively minute and difficult to cauterize without damaging the mouthparts (Felton lab, personal observations). Pharmacological techniques include directly treating plant tissues with OS or salivary secretions. Typically OS is collected by inducing larvae to regurgitate which is then applied to artificial plant wounds. Saliva has been prepared by surgical removal and homogenization of salivary glands with subsequent assay of their biological and/or chemical properties. These techniques have been widely used to study salivary gland components in blood-feeding arthropods (Valenzuela 2002; Ribeiro et al. 2004) and in herbivorous insects (Musser et al. 2002; Shackel et al. 2005). Unfortunately, in many cases it has not been determined which components of OS or salivary gland homogenates are normally secreted during feeding. Without further studies showing that these components are actually deposited during feeding, pharmacological studies are of restricted value. Fortunately, several approaches are available to identify secreted components. First, collection of saliva can be made directly from the spinneret (Liu et al. 2004) or saliva that is deposited on a feeding substrate can be collected and analyzed. The limitation of this approach is that at present only very minute quantities (ca.

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